CCL20 as a predictor of clinical response to IL23-antagonists

ABSTRACT

The present invention relates to the use of the Chemokine (C—C motif) ligand 20 (CCL20) as a biomarker to stratify or identify populations of patients suffering from interleukin-23 (IL23)-mediated diseases (e.g., Crohn&#39;s disease) responsive to treatment with an, anti-IL23 antagonist (including, e.g., anti-IL23 antibodies). Levels of CCL20 above or below a predetermined threshold can be used, for example, (i) to determine whether a patient with an IL23-mediated disease or disorder such a Crohn&#39;s disease is eligible or non-eligible for treatment with a therapeutic agent, (ii) to determine whether treatment with a certain agent should be commenced, suspended, or modified, (iii) to diagnose whether the IL23-mediated disease is treatable or not treatable with a specific therapeutic agent, or (iv) to predict the outcome of treating the IL23-mediated disease with a specific therapeutic agent. CCL20 can be used in combination with other IL23 pathway biomarkers such as IL22 and/or lipocalin-2 (LCN2).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase of PCT/US2016/067120, filedDec. 16, 2016, which claims priority to U.S. Provisional PatentApplication No. 62/271,156, filed Dec. 22, 2015, which are incorporatedherein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name: 50315PCT_SequenceListing.txt; Size: 72,947 bytes; andDate of Creation: Dec. 16, 2016) filed with the application isincorporated herein by reference in its entirety.

FIELD

The present disclosure relates to the use of expression levels of theChemokine (C—C motif) ligand 20 (CCL20) cytokine to stratify and/oridentify populations of patients having an IL23-mediated disease ordisorder suitable for treatment with an IL23 antagonist (including,e.g., an anti-IL23 antibody or fragment thereof).

BACKGROUND

Interleukin (IL)-23 is a proinflammatory cytokine implicated in thepathogenesis of various inflammatory conditions including but notlimited to Crohn's disease (CD), ulcerative colitis (UC), psoriasis,psoriatic arthritis, rheumatoid arthritis, and ankylosing spondylitis.IL23 induces T-cells to express a number of inflammatory genes includingIL17A, IL17A receptor, TNF-α, and GM-CSF. The main known effects of IL23are to drive the differentiation of T helper Th17 cells, as well asmacrophages, natural killer (NK) cells, dendritic cells, and innatelymphoid cells leading to up-regulation of IL17, IL22, TNFα, GMCSF, andIFN-γ, and down-regulation of IL10.

Interleukin (IL)-23 is a heterodimeric cytokine consisting of 2subunits: p40 and p19. The p40 subunit is shared by IL12 and IL23 as acommon subunit, and is targeted by inhibitors of IL12/23, e.g.,ustekinumab/STELARA® (Janssen Biotech, Inc, Horsham, Pa.) andbriakinumab/ABT-874 (Abbott Laboratories, Abbott Park, Ill.).

Studies in patients have demonstrated that IL23 is upregulated in cellsand target tissues of Crohn's disease (CD) and ulcerative colitis (UC),while IL12 is not (Schmidt et al. Inflamm Bowel Dis. 11(1):16-23(2005)). Similar observations have been reported in dendritic cells frompatients with multiple sclerosis (MS; Vaknin et al. J of Immunol.176:7768 74 (2006)), patients with psoriasis (Lee et al J Exp Med199:125-30 (2004)), and active lesions from patients with MS (Li et al.Brain. 130(2):490-501 (2007)). IL23 is also elevated in other diseasesincluding rheumatoid arthritis (RA), ankylosing spondylitis (AS),chronic obstructive pulmonary disease (COPD), and neuromyelitis optica.Genome-wide association studies in CD and psoriasis (PsO) patientsshowed significant association between polymorphisms in the unique IL23receptor component (IL23R) and disease (Cargill et al. Am J of HumanGen. 80:273-90 (2007); Duerr et al. Science 314:1461-63 (2006)).Furthermore, allelic variants of IL23R have shown significantcorrelation with the frequency of UC (Cargill et al. Am. J. Hum. Gen.80:273-90 (2007)), RA (Farago et al. Ann. Rheum. Dis. 67:248-50 (2008)),AS (Burton et al. Nature Gen. 39:1329-37 (2007)), and MS (Illes et al.Neuro Letters. 431:36-38 (2008)). Therefore, both IL23 and its receptorare very attractive targets for drug development.

In preclinical models of inflammatory bowel disease (IBD) (Ahern et al.Immun Rev. 226:147-59 (2008)), PsO inflammatory arthritis (Yago et al.Arthritis Res and Ther. 9:R96 (2007)), and MS (Cua et al. Nature421:744-48 (2003), the beneficial effects of anti-IL12/23p40 antibodieshave been recapitulated through the blockade of IL23 alone while sparingIL12. In the clinic, anti-IL12/23p40 antibodies (e.g., ustekinumab andbriakinumab) have been shown to induce clinical responses in CD (Phase 2studies; Toedter et al. Am J Gastroenterol. 104(11):2768-73 (2009);Sandborn et al. Gastroenterol. 135:1130-41 (2008); Mannon et al. N Eng Jof Med. 351(20):2069-79 (2004)) and PsO (Phase 2 and 3 studies; Gordonet al. J. Invest. Dermatol. 132:304-314 (2012); Kimball et al. Br JDermatol. 167(Suppl. 1):64 (Abstract P94) (2012); Langley et al. J AmAcad Dermatol. 66:AB195 (Abstract 4779) (2012); Gottlieb et al. Br. J.Dermatol. 165:652-660 (2011); Reich et al. N Engl J Med. 365:1586-1596(2011); Strober et al. Br J Dermatol. 165:661-668 (2011); Leonardi etal. Lancet. 371(9625):1665-74 (2008); Papp et al. Lancet.371(9625):1675-84 (2008)). Phase 1 clinical studies with anti-IL23antibodies MEDI2070 (NCT01094093) and CNTO 1959 (Sofen et al. DrugsDermatol. 10(8):885-92 (2011)) in subjects with PsO have demonstratedclinical efficacy comparable with antibodies targeting IL12 and IL23indicating that therapeutic effects of the anti-IL12/23p40 antibodiesmay be due to neutralization of IL23 alone (Papp et al. Efficacy andSafety of Different Dose Regimens of a Selective IL-23p19 Inhibitor (BI655066) Compared with Ustekinumab in Patients with Moderate-to-SeverePlaque Psoriasis with and without Psoriatic Arthritis [abstract]. In:2015 ACR/ARHP Annual Meeting. Abstract nr 2144).

Although several agents targeting different elements of the IL23 pathwayare currently approved or under development, the morbidity,complications and intrinsic heterogeneity of IL23-mediated diseasescontinue to demand new therapies and/or improved methods of identifyingspecific populations of patients suitable for treatment with IL23antagonists. See, e.g., Gaffe et al. Nature Revs. Immunol. 14:585-600(2014).

BRIEF SUMMARY

The present disclosure provides a method of treating an interleukin-23(IL23)-mediated disease in a patient, comprising administering an IL23antagonist to a patient if the patient is determined to have (i) a loweror decreased level of CCL20 in one or more samples taken from thepatient compared to a predetermined CCL20 threshold level, or comparedto a CCL20 level in one or more control samples. Also provided is methodof treating a patient having an IL23-mediated disease comprisingsuspending or not initiating the administration of an IL23 antagonist toa patient if the patient is determined to have a higher or increasedlevel of CCL20 in one or more samples taken from the patient compared toa predetermined CCL20 threshold level, or compared to a CCL20 level inone or more control samples.

Also provided is method of treating an interleukin-23 (IL23)-mediateddisease in a patient, wherein the patient failed, was non-responsive orintolerant to treatment with an anti-TNF agent comprising administeringan IL23 antagonist to the patient if the patient is determined to have alower or decreased level of CCL20 in one or more samples taken from thepatient compared to a predetermined CCL20 threshold level, or comparedto a CCL20 level in one or more control samples.

The disclosure provides also a method of determining whether to treat apatient having an IL23-mediated disease with an IL-23 antagonist,comprising determining to treat the patient if the patient is determinedto have a lower or decreased level of CCL20 in one or more samples takenfrom the patient compared to a predetermined CCL20 threshold level, orcompared to a CCL20 level in one or more control samples. Also providedis a method of selecting a patient diagnosed with an IL23-mediateddisease as a candidate for treatment with an IL23 antagonist, comprisingselecting the patient for treatment if the patient is determined to havelower or decreased level of CCL20 in one or more samples taken from thepatient compared to a predetermined CCL20 threshold level, or comparedto a CCL20 level in one or more control samples.

In some aspects, the methods disclosed above further comprise measuringthe level of CCL20 in one or more of the samples obtained from thepatient or instructing a clinical laboratory or healthcare provider tomeasure the level of CCL20 in the sample and/or submitting the one ormore samples obtained from the patient to a clinical laboratory orhealthcare provider to measure the level of CCL20 in the sample. In someaspects, the methods disclosed above further comprise determining thelevel of CCL20 in the one or more samples obtained from the patient. Insome aspects, the methods disclosed above further comprise advising ahealthcare provider to

(i) administer an IL23 antagonist to the patient if the patient isdetermined to have a lower or decreased level of CCL20 in one or more ofthe samples compared to a predetermined CCL20 threshold level, orcompared to a CCL20 level in one or more control samples, or to(ii) suspend or deny the administration of an IL23 antagonist to thepatient if the patient is determined to have a higher or increased levelof CCL20 in one or more of the samples compared to a predetermined CCL20threshold level, or compared to a CCL20 level in one or more controlsamples.

In some aspects, the second sample is taken 1, 2, 4, 8, 12, or 28 weeks,or at intervening times, after administering the IL23 antagonist. Insome aspects, the IL23 antagonist is an anti-IL23 antibody orantigen-binding fragment thereof. In some aspects, the anti-IL23antibody or antigen-binding fragment thereof binds to the p19 subunit ofIL23 (SEQ ID NO: 15), to the p40 subunit of IL23 (SEQ ID NO: 16), orboth.

In some aspects, the anti-IL23 antibody or antigen-binding fragmentthereof comprises ustekinumab, briakinumab, guselkumab, BI-655066,tildrakinumab, LY-3074828, or an antigen-binding fragment thereof. Insome aspects, the anti-IL23 antibody or antigen-binding fragment thereofcomprises (i) a variable region (VH) comprising or consisting of SEQ IDNO: 7 and/or a light chain variable region (VL) comprising or consistingof SEQ ID NO: 8, or (ii) a variable region (VH) comprising or consistingof SEQ ID NO: 45 and/or a light chain variable region (VL) comprising orconsisting of SEQ ID NO: 46. In some aspects, the anti-IL23 antibody orantigen-binding fragment thereof comprises at least one complementaritydetermining region selected from SEQ ID NOS: 33-38 or SEQ ID NOS: 47-52.In some aspects, the antibody is administered at a fixed dose. In someaspects, the fixed dose is between 10 and 1000 mg/dose. In some aspects,the fixed dose is about 210 mg/dose or about 700 mg/dose.

In some aspects, the patient has been treated before, during, after, oralternatively to the administration of an IL23 antagonist or anti-IL23antibody or antigen-binding fragment with one or more additionaltherapies for the treatment of the IL23-mediated disease or disorder. Insome aspects, the one or more samples taken from the patient and/or theone or more control samples are one or more selected from the groupconsisting of: whole blood, blood serum, plasma, saliva, sputum,bronchoalveolar lavage fluid, cerebrospinal fluid, pleural fluid,pericardial fluid, ascites, synovial fluid, epithelial cells, urine,stool, skin, tissue biopsy, or a combination thereof.

In some aspects, the one or more control samples are (i) a sample orsamples obtained from normal healthy individuals; (ii) a sample orsamples obtained from patients with a non-IL23-mediated disease; or(iii) a combination thereof. In some aspects, the patient's level ofCCL20 is measured in an immunoassay.

In some aspects, the methods disclosed above further comprisedetermining the level of one or more IL23 Pathway Biomarkers selectedfrom the group consisting of IL22, LCN2, IL17F, IL17A/F, IL23R, IL12B,IL6, IL21, TNF, CCR6, CCL22, IL1R1, IFN-γ, S100A12, DEFB-2, DEFB-4, Ill,SERPINB3, PI3/Elafin, LL37, RORγ, RORγT, IL26, S100A7, DEFB103B, andGM-CSF. In some aspects, the predetermined threshold level of CCL20 isselected from the group consisting of:

(a) about the mean level of CCL20;

(b) about the median level of CCL20; and

(c) about the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, or 9th decilebaseline level of CCL20 as described in TABLE 6;

as measured in the serum using an immunoassay from a plurality of normalhealthy patients, patients with a non-IL23-mediated disease, and/orpatients with an IL23-mediated disease.

In some aspects, the IL23-mediated disease or disorder is a pulmonarydisease, an inflammatory bowel disease, a chronic inflammatory skindisease, an inflammatory disease, an autoimmune disease, aneurodegenerative disease, an infection, or a cancer. In some aspects,the IL23-mediated disease or disorder is selected from the groupconsisting of: asthma, IPF, COPD. Crohn's disease, ulcerative colitis(UC), celiac disease, atopic dermatitis, allergic contact dermatitis,eczema, psoriasis, alopecia areata, palmoplantar pustulosis, psoriaticarthritis, anklyosing spondylitis, arthritis, rheumatoid arthritis (RA),a rheumatic disorder, ANCA vasculitis, Bechet's disease, autoimmunethyroiditis, type 1 diabetes, multiple sclerosis (MS), Sjogren'ssyndrome (SS), systemic lupus erythematosus (SLE), Alzheimer's disease,mycobacterial disease, leishmaniasis, a fungal infection, a viralinfection, gastric cancer, colorectal cancer, esophageal cancer,leukemia, hepatitis B virus (HBV)-related hepatocellular carcinoma,breast cancer, lung cancer, and nasopharyngeal cancer. In some aspects,the inflammatory bowel disease is Crohn's disease, UC or Celiac Disease.

In some aspects, the patient is determined to have a level of CRP≥5 mg/Land/or a level of FCP≥250 μg/g, a level of FCP≥200 μg/g, a level ofFCP≥150 μg/g, a level of FCP≥100 μg/g, or a level of FCP at least about100 μg/g to at least about 250 μg/g in one or more samples taken fromthe patient. In some aspects, the predetermined CCL20 threshold level isat least about 5 pg/mL to at least about 55 pg/mL as measured using animmunoassay. In some aspects, the predetermined CCL20 threshold level isabout 22.6 pg/mL as measured using an immunoassay. In some aspects,administration of the IL23 antagonist or anti-IL23 antibody orantigen-binding fragment thereof results in a Crohn's Disease ActivityIndex (CDAI) response score reduction of at least 100 points and/or areduction in the total CDAI score to below 150 points after firstadministering the anti-IL23 antibody or antigen-binding fragmentthereof. In some aspects, the CDAI response score reduction of at least100 points or reduction in the total CDAI score to below 150 pointsoccurs within 1, 2, 4, 8, 12, 16 or 24 weeks or later after firstadministering the IL23 antagonist or anti-IL23 antibody orantigen-binding fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows patient disposition up to Week 24. The chart summarizes thenumber and disposition of enrolled and randomized patients in a phase 2astudy (clinicaltrials.gov identifier: NCT01714726) evaluating MEDI2070in patients having moderate or severe CD. mITT, Modified Intent-to-TreatPopulation; PP, per protocol; OL, Open Label.

FIG. 2A shows clinical efficacy at Week 8 in the ModifiedIntent-to-Treat Population as indicated by CDAI clinical effect, CDAIremission, and CR100. The rate of CDAI clinical effect (defined as aCDAI score less than 150 or reduction from baseline in CDAI score of atleast 100 points) at week 8 was significantly higher in the MEDI2070group versus the placebo group (49.2% vs. 26.7%, respectively; P=0.01).CDAI remission (defined as a CDAI score less than 150) rates were 27.1%with MEDI2070 and 15.0% with placebo (P=0.10). CR100 rates (defined asat least a 100-point CDAI score reduction) were 45.8% and 25.0% in theMEDI2070 and placebo groups, respectively (P=0.02).

FIG. 2B shows clinical efficacy at Week 8 in the ModifiedIntent-to-Treat Population as indicated by composite end points. Asignificantly greater proportion of patients receiving MEDI2070 achievedthe composite end points of CDAI effect and 50% reduction in FCP or CRPversus baseline (P<0.001), and CDAI remission and 50% reduction in FCPor CRP versus baseline (P=0.02). CDAI, Crohn's Disease Activity Index;CI, confidence interval; CRP, C-reactive protein; FCP, fecalcalprotectin; p, p-value.

FIG. 3A shows efficacy at various time points through Week 24 in theOpen-Label Population using nonresponder imputation as indicated byCrohn's Disease Activity Index effect rate (from baseline to week 24).Sample size of open-label period: placebo/MEDI2070 group (n=52);MEDI2070 group (n=52).

FIG. 3B shows efficacy at various time points through Week 24 in theopen-label population using nonresponder imputation as indicated byCrohn's Disease Activity Index remission rate (from baseline to week24). Sample size of open-label period: placebo/MEDI2070 group (n=52);MEDI2070 group (n=52).

FIG. 4 shows percent of patients with CDAI-100 response over time bybaseline CCL20 Levels. CCL20 LO, baseline CCL20<22.6 pg/mL; CCL20 HI,baseline CCL20≥22.6 pg/mL. CI, confidence interval. Median CCL20concentration across study population: 22.6 pg/mL. Patients withbaseline serum CCL20 level<22.6 pg/mL had a statistically significantincreased CDAI-100 response compared to placebo at week 8.

FIG. 5 shows percent of patients with CDAI-100 response over time bybaseline IL22 levels. IL22 LO, baseline IL22<15.6 pg/mL; IL22 HI,baseline IL22≥15.6 pg/mL. CI, confidence interval. Median IL22concentration across study population: 15.6 pg/mL. 116 patients hadevaluable baseline IL22 values. Patients with baseline serum IL22levels≥15.6 pg/mL had a statistically significant increased CDAI-100response compared to placebo at week 8.

FIG. 6 shows percent of patients with CDAI-100 response over time bybaseline LCN2 Levels. LCN2 LO, baseline LCN2<215 ng/mL; LCN2 HI,baseline LCN2≥215 ng/mL. CI, confidence interval. Median LCN2concentration across study population: 215 ng/mL. Patients with baselineserum LCN2 levels≥215 ng/mL had a statistically significant increasedCDAI-100 response compared to placebo at week 8.

FIG. 7 shows the differential clinical response rate between subjectstreated with MEDI2070 compared to placebo as measured by the differencebetween the percentage (%) of subjects achieving a CDAI score<150 or areduction in CDAI score of >100) at Week 8 as a function of baselineCCL20, IL22 or LCN2 levels (by deciles). For the set of baseline valuesof CCL20, IL22 or LCN2 across the study population in each of FIGS. 7-9,each distribution was divided into 10 levels, or deciles, such that eachof the 11 analyte cut-offs (noted as “quantile” in FIGS. 7-9)progressively segmented the study population into groups with 10% lessof the total study population. For example, at 4^(th) decile (shown asthe 0.4 quantile), 40% of the total study population had a baselineCCL20, IL22 or LCN2 serum level less than a particular analyte levelwhile 60% of the total study population had a baseline CCL20, IL22 orLCN2 serum level greater than or equal to the particular analyte level.

FIG. 8 shows the differential clinical response rate between subjectstreated with MEDI2070 compared to placebo as measured by the differencebetween the percentage (%) of subjects achieving a 100 point improvementin CDAI score at Week 8 as a function of baseline CCL20, IL22, or LCN2levels (by deciles).

FIG. 9 shows the differential clinical response rate between subjectstreated with MEDI2070 compared to placebo as measured by the differencebetween the percentage (%) of subjects achieving a CDAI score<150 or areduction in CDAI score of >100, + also achieving a >50% reduction ineither FCP or CRP compared to baseline FCP or CRP; between subjectstreated with MEDI2070 and placebo at Week 8 as a function of baselineCCL20, IL22 and LCN2 levels (by deciles). Clinical Response rates arereported as the delta or difference between the rate observed forMEDI2070 treated subjects and the rate observed for subjects treatedwith placebo (“Response Rate Differential”).

As shown in FIGS. 7-9, CD patients treated with MEDI2070 havingincreasingly higher levels of baseline IL22 or LCN2 achieved higherclinical response rates (as measured using three different clinicalresponse measurements) compared to placebo, illustrating that MEDI2070induced better clinical responses in patients with high baseline IL22 orLCN2 serum levels at week 8. Subjects with high levels of IL22 or LCN2(including, e.g., subjects with IL22 or LCN2 levels at the 5^(th),6^(th) or 7^(th) deciles (0.5, 0.6 or 0.7 quantiles)) had greaterclinical response rate differences from placebo (irrespective of whichof the three different clinical response measurements was used) comparedto the IL22 or LCN2 low subjects (including, e.g. subjects with IL22 orLCN2 levels at the 1^(st) or 2^(nd) deciles (0.1 or 0.2 quantiles)). CDpatients treated with MEDI2070 having increasingly lower levels ofbaseline CCL20 achieved higher clinical response rates (as measuredusing three different clinical response measurements) compared toplacebo, illustrating that MEDI2070 induced better clinical responses inpatients with low baseline CCL20 serum levels at week 8. Subjects withlow levels of CCL20 (including, e.g., subjects with CCL20 levels at the1^(st) or 2^(nd) deciles (0.1 or 0.2 quantiles)) had greater clinicalresponse rate differences from placebo (irrespective of which of thethree different clinical response measurements was used) compared to theCCL20 high subjects (including, e.g. subjects with CCL20 at the 7^(th)or 8^(th) deciles (0.7 or 0.8 quantiles)). These results underscore thepredictive value of high or elevated IL22 and/or LCN2 serum levels, andlow or decreased CCL20 serum levels, in identifying patients responsiveto treatment with MEDI2070. A summary of the baseline CCL20, IL22 orLCN2 serum levels corresponding to each of the deciles described inFIGS. 7-9 is provided in Example 4 (see TABLE 4 (IL22), TABLE 5 (LCN2)or TABLE 6 (CCL20)).

FIG. 10 compares the differential clinical response rate (defined as thedifference in the percentage (%) of subjects achieving a CDAI-100response between treatment and placebo (“CDAI Response Delta vs.Placebo”)) (y axis) and the differential clinical remission rate(defined as the difference in the percentage (%) of subjects achieving atotal CDAI score of below 150 points between treatment and placebo(“CDAI Remission Delta vs. Placebo”)) (x axis) for patients treated withvarious molecules administered for 4-10 weeks as shown. Patients treatedwith MEDI2070 for 8 weeks who had a baseline CRP≥5 mg/L (n=85); baselineIL-22≥11.3 pg/mL (n=81); baseline IL-22≥15.6 pg/mL (n=58); or baselineIL-22≥11.3 pg/mL+CRP≥5 mg/L (n=62) (as measured using the IL22immunoassay described in Example 3) had increased CDAI-100 responserates and/or CDAI remission rates. Both the CDAI-100 response rates andthe CDAI remission rates of patients treated with MEDI2070 for 8 weekswho had a baseline CRP≥5 mg/L; baseline IL-22≥11.3 pg/mL; baselineIL-22≥15.6 pg/mL; or baseline IL-22≥11.3 pg/mL+CRP≥5 mg/L (as measuredusing the IL22 immunoassay described in Example 4) were greater than thereported CDAI-100 response rates and/or the CDAI remission rates forother molecules currently approved or under development to treat CDpatients including: Ustekinumab (after 6 weeks or 8 weeks of treatmentwith a 6 mg/kg dose as reported in FIG. 1 of Sandborn et al., N Engl JMed. 2012 Oct. 18; 367(16):1519-28.); Vedolizumab (after 6 weeks or 10weeks of treatment as reported in FIG. 3 of Sands et. al.,Gastroenterology. 2014 September; 147(3):618-627); or Adalimumab (after4 weeks of treatment in patients who are secondary failures toinfliximab as reported in Sandborn et. al, Ann Intern Med. 2007;146:829-838). The overall clinical response and clinical remission ratesfor all patients treated with MEDI2070 in the Phase 2a study,irrespective of biomarker status (mITT (n=119)), was similar to theresponse rates of other molecules currently approved or underdevelopment. mITT, Modified Intent-to-Treat Population. TABLE 6summarizes the CDAI-100 response rate differential and the CDAIremission rate differential for each of the MEDI2070-treated subgroupsplotted in FIG. 10.

DETAILED DESCRIPTION

The present disclosure relates to the use of chemokine (CC motif) ligand20 (CCL20), alone or in combination with one or more biomarkers, e.g.,interleukin-22 (IL22), lipocalin-2 (LCN2), C-reactive protein (CRP), orfecal calprotectin (FCP), to predict clinical outcomes in patientssuffering from interleukin 23 (IL23)-mediated diseases and treated withan IL23 antagonist which target, for example, the p40 subunit of IL12and IL23 (targeted, e.g., by ustekinumab), or the p19 subunit of IL23(targeted, e.g., by tildrakinumab, guselkumab, MEDI2070, BI-655066, andLY-3074828).

Genes or proteins in the IL23 pathway that can be used as biomarkers incombination with CCL20 according to the methods disclosed hereininclude, e.g., interleukin-22 (IL22), lipocalin-2 (LCN2), IL23R, IL12B,IL6, IL21, TNF, CCR6, CCL22, IL1R1, IFN gamma, S100 calcium-bindingprotein A12 (S100A12), defensin B2 (DEFB-2, DEFB-4), interleukin-1 beta(IL1(3), serine (or cysteine) proteinase inhibitor member 3 (SERPINB3),peptidase inhibitor 3 (PI3)/Elafin, human cathelicidin (LL37),retinoid-related orphan receptor-γ (RORy and RORγT), interleukin-26(IL26), psoriasin (S100A7), defensin beta 103B (DEFB103B), or GM-CSF.See Haider et al. J. Immunol. 180: 1913-1920 (2008); Nakae et al. J.Leukocyte Biol. 81: 1258-1268 (2007); Guttman-Yassky et al. J Immunol.181(10): 7420-7427 (2008); El-Behi Nature Immunol. 12:568-75 (2011),Wilson, et al., Nature Immunology 8:950-7 (2007); all of which areherein incorporated by reference in their entireties.

The present disclosure provides, for example, methods to treat, preventand/or ameliorate an IL23-mediated disease, to predict clinicaloutcomes, select patients for treatment, or stratify a population ofpatients suffering from a specific disease or disorder mediated by IL23based on determining the expression level of CCL20 alone, or incombination with the expression level of one, two, three, or morebiomarkers, including, e.g., IL22, LCN2, CRP, and FCP. Accordingly, inone aspect, the present disclosure provides methods comprisingdetermining the expression level of CCL20, alone or in combination withone or more biomarkers (e.g., levels of IL22, levels of LCN2, levels ofFCP, levels or CRP, or combinations thereof) in a sample obtained from asubject having an IL23-mediated disease to determine the appropriatecourse of treatment.

CD is a chronic transmural inflammatory disease of unknown etiology thatmost commonly affects the distal ileum and colon, and may occur in anypart of the gastrointestinal (GI) tract. Patients with CD haveuncontrolled inflammation that causes direct or collateral damage to theintestinal mucosa. The leading current hypothesis is that, ingenetically predisposed individuals, this inflammation can result eitherfrom persistence of inflammatory stimulus, due to impaired gut barrierfunction, or from a dysregulated inflammatory response (Sandborn et al.Gastroenterol. 135:1130-41 (2008); Rutgeerts et al. Aliment PharmacolTher. 17(12):1435-50 (2003)). CD occurs most commonly between the agesof 15 and 35 years, although patients of any age may be affected.

Commonly used medical therapies include aminosalicylates, (includingsulfasalazine and mesalamine), systemic corticosteroids,immunosuppressive agents (e.g., azathioprine and methotrexate);antibacterial agents; and biologic agent, e.g., adalimumab/HUMIRA®(Abbott laboratories, IL) (SEQ ID NOS: 23, 24), infliximab/REMICADE®(Janssen Biotech, Inc), certolizumab/CIMZIA® (UCB, Inc, Smyrna, Ga.)(SEQ ID NOS: 25, 26), vedolizumab/ENTYVIO® (Takeda PharmaceuticalsAmerica, Inc., Deerfield, Ill.), and Natalizumab/TYSABRI™ (Biogen IdecInc, Cambridge, Mass.). Being a highly heterogeneous disease, it isdifficult to predict whether a patient will respond favorably to acertain therapy.

The biomarkers disclosed herein (e.g., an expression range and/orthreshold level of CCL20, alone or in combination with, for example,elevated or high levels of clinical biomarkers IL22, LCN2, CRP, FCP, andcombinations thereof), can be used to stratify and/or identify apopulation of subjects having an IL23-mediated disease such as CDsuitable for treatment with an IL23 antagonists (including, e.g., ananti-IL23 antibody or antigen-binding fragment thereof).

Within each one of the strata, the biomarkers disclosed herein (e.g., anexpression range and/or threshold level of CCL20, alone or incombination with, for example, elevated or high levels of clinicalbiomarkers IL22, LCN2, CRP, FCP, and combinations thereof), can be used,for example, for diagnosing a patient, treating a patient, for example,with an IL23 antagonist (e.g., an anti-IL23 antibody targeting, e.g.,the p19 subunit of IL23); selecting or non-selecting a patient fortreatment, for example, with an IL23 antagonist (e.g., an anti-IL23antibody targeting, e.g., the p19 subunit of IL23); selecting a certaintreatment, for example, with an IL23 antagonist (e.g., an anti-IL23antibody targeting, e.g., the p19 subunit of IL23); suspending ormodifying temporarily or permanently a treatment; determining theprognosis of a patient; or monitoring the effect of a treatment, whereinthose methods comprise, for example, determining an expression rangeand/or threshold level of CCL20, alone or in combination with, forexample, elevated or high levels of clinical biomarkers IL22, LCN2, CRP,FCP, and combinations thereof.

In some aspects, the IL23 antagonist is an anti-IL23 antibody which canspecifically bind to the p19 subunit of IL23 (SEQ ID NO: 15), to the p40subunit of IL23 (SEQ ID NO: 16), or both. In some aspects, the IL23antagonist is an anti-IL23 antibody or other molecule (e.g., smallmolecule, aptamer, scaffolding molecule, etc) which can compete forbinding to the p19 subunit of IL23 (SEQ ID NO: 15), to the p40 subunitof IL23 (SEQ ID NO: 16), or both, with MEDI2070 or another anti-IL23antibody known in the art.

In some aspects, the anti-IL23 antibody or antigen-binding fragmentthereof comprises the heavy chain (HC) (SEQ ID NO: 17) and/or the lightchain (SEQ ID NO: 18) of MEDI2070, or an antigen-binding fragment,variant, or derivative thereof. In some aspects, the anti-IL23 antibodyor antigen-binding fragment thereof comprises the variable region (VH)(SEQ ID NO: 7) and/or the light chain variable region (VL) (SEQ ID NO:8) of MEDI2070. In some aspects, the anti-IL23 antibody orantigen-binding fragment thereof comprises at least one of thecomplementarity determining regions of MEDI2070 (SEQ ID NOS: 33-38). Insome aspects, the anti-IL23 antibody or antigen-binding fragment thereofcomprises a VH region comprising the sequence of SEQ ID NO: 45 and/or aVL region comprising the sequence of SEQ ID NO: 46, or anantigen-binding fragment, variant, or derivative thereof. In someaspects, the anti-IL23 antibody or antigen-binding fragment thereofcomprises at least one of the complementarity determining regions of SEQID NOS: 47-49 (CDRs of the VH of SEQ ID NO: 45) and/or SEQ ID NOS: 50-52(CDRs of the VL of SEQ ID NO:46).

In other aspects, the IL23 antagonist is an anti-IL23 antibody selectedfrom ustekinumab or briakinumab (targeting the p40 subunit of IL23),guselkumab, tildrakizumab, BI-655066 or LY-3074828 (targeting the p19subunit of IL23), an antigen-binding fragment thereof, or a combinationthereof. In other aspects, the IL23 antagonist is a molecule competingwith ustekinumab or briakinumab (targeting the p40 subunit of IL23),guselkumab, tildrakizumab, BI-655066 or LY-3074828 (targeting the p19subunit of IL23), an antigen-binding fragment thereof, or a combinationthereof for binding to IL23.

The methods disclosed herein are not only applicable to IL23-bindingantibodies. The method disclosed herein can be applied to any inhibitorsof the p19 subunit of IL23, including for example antibodies andantigen-binding fragments thereof, aptamers, scaffolding molecules,small molecules, soluble IL23 receptors, etc.

In order that the present disclosure can be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

I. Definitions

The headings provided herein are not limitations of the various aspectsor aspects of the disclosure, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

In this specification and the appended claims, the singular forms “a”,“an” and “the” include plural referents unless the context clearlydictates otherwise. The terms “a” (or “an”), as well as the terms “oneor more,” and “at least one” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

Wherever aspects are described herein with the language “comprising,”otherwise analogous aspects described in terms of “consisting of” and/or“consisting essentially of” are also provided.

The term “about” as used in connection with a numerical value throughoutthe specification and the claims denotes an interval of accuracy,familiar and acceptable to a person skilled in the art. In general, suchinterval of accuracy is ±15%.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form.

Numeric ranges are inclusive of the numbers defining the range. Where arange of values is recited, it is to be understood that each interveninginteger value, and each fraction thereof, between the recited upper andlower limits of that range is also specifically disclosed, along witheach subrange between such values. The upper and lower limits of anyrange can independently be included in or excluded from the range, andeach range where either, neither or both limits are included is alsoencompassed within the invention. Where a value is explicitly recited,it is to be understood that values which are about the same quantity oramount as the recited value are also within the scope of the invention.Where a combination is disclosed, each subcombination of the elements ofthat combination is also specifically disclosed and is within the scopeof the invention. Conversely, where different elements or groups ofelements are individually disclosed, combinations thereof are alsodisclosed. Where any element of an invention is disclosed as having aplurality of alternatives, examples of that invention in which eachalternative is excluded singly or in any combination with the otheralternatives are also hereby disclosed; more than one element of aninvention can have such exclusions, and all combinations of elementshaving such exclusions are hereby disclosed.

Amino acids are referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Unless otherwise indicated, aminoacid sequences are written left to right in amino to carboxyorientation.

Nucleotides are referred to by their commonly accepted single-lettercodes. Unless otherwise indicated, nucleic acids are written left toright in 5′ to 3′ orientation. Nucleotides are referred to herein bytheir commonly known one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Accordingly, A represents adenine,C represents cytosine, G represents guanine, T represents thymine, Urepresents uracil.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid,” “nucleicacid molecule,” and “gene” are used interchangeably herein to refer topolymers of nucleotides of any length, and ribonucleotides,deoxyribonucleotides, analogs thereof, or mixtures thereof.

The phrase “DNA sequence” refers to a contiguous nucleic acid sequence.The sequence can be either single stranded or double stranded, DNA orRNA, but double stranded DNA sequences are preferable. The sequence canbe an oligonucleotide of 6 to 20 nucleotides in length to a full lengthgenomic sequence of thousands or hundreds of thousands of base pairs.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer can be linear or branched, it can comprise modifiedamino acids, and it can be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids such as homocysteine, ornithine, p-acetylphenylalanine,D-amino acids, and creatine), as well as other modifications known inthe art.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, polynucleotide, or compositionwhich is in a form not found in nature. Isolated polypeptides,polynucleotides, or compositions include those which have been purifiedto a degree that they are no longer in a form in which they are found innature. In some aspects, a polypeptide, polynucleotide, or compositionwhich is isolated is substantially pure.

The term “amino acid substitution” refers to replacing an amino acidresidue present in a parent sequence with another amino acid residue. Anamino acid can be substituted in a parent sequence, for example, viachemical peptide synthesis or through recombinant methods known in theart. Accordingly, references to a “substitution at position X” or“substitution at position X” refer to the substitution of an amino acidpresent at position X with an alternative amino acid residue. In someaspects, substitution patterns can described according to the schemaAXY, wherein A is the single letter code corresponding to the amino acidnaturally present at position X, and Y is the substituting amino acidresidue. In other aspects, substitution patterns can described accordingto the schema XY, wherein Y is the single letter code corresponding tothe amino acid residue substituting the amino acid naturally present atposition X.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, if an amino acid in apolypeptide is replaced with another amino acid from the same side chainfamily, the substitution is considered to be conservative. In anotheraspect, a string of amino acids can be conservatively replaced with astructurally similar string that differs in order and/or composition ofside chain family members.

Non-conservative substitutions include those in which (i) a residuehaving an electropositive side chain (e.g., Arg, His or Lys) issubstituted for, or by, an electronegative residue (e.g., Glu or Asp),(ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by,a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val), (iii) acysteine or proline is substituted for, or by, any other residue, or(iv) a residue having a bulky hydrophobic or aromatic side chain (e.g.,Val, His, Ile or Trp) is substituted for, or by, one having a smallerside chain (e.g., Ala, Ser) or no side chain (e.g., Gly).

Other substitutions can be readily identified by workers of ordinaryskill. For example, for the amino acid alanine, a substitution can betaken from any one of D-alanine, glycine, beta-alanine, L-cysteine andD-cysteine. For lysine, a replacement can be any one of D-lysine,arginine, D-arginine, homo-arginine, methionine, D-methionine, omithine,or D-ornithine. Generally, substitutions in functionally importantregions that can be expected to induce changes in the properties ofisolated polypeptides are those in which (i) a polar residue, e.g.,serine or threonine, is substituted for (or by) a hydrophobic residue,e.g., leucine, isoleucine, phenylalanine, or alanine; (ii) a cysteineresidue is substituted for (or by) any other residue; (iii) a residuehaving an electropositive side chain, e.g., lysine, arginine orhistidine, is substituted for (or by) a residue having anelectronegative side chain, e.g., glutamic acid or aspartic acid; or(iv) a residue having a bulky side chain, e.g., phenylalanine, issubstituted for (or by) one not having such a side chain, e.g., glycine.The likelihood that one of the foregoing non-conservative substitutionscan alter functional properties of the protein is also correlated to theposition of the substitution with respect to functionally importantregions of the protein: some non-conservative substitutions canaccordingly have little or no effect on biological properties.

The term “amino acid insertion” refers to introducing a new amino acidresidue between two amino acid residues present in the parent sequence.An amino acid can be inserted in a parent sequence, for example, viachemical peptide synthesis or through recombinant methods known in theart. Accordingly as used herein, the phrase “insertion between positionsX and Y” wherein X and Y correspond to amino acid positions, refers tothe insertion of an amino acid between the X and Y positions, and alsoto the insertion in a nucleic acid sequence of a codon encoding an aminoacid between the codons encoding the amino acids at positions X and Y.

The term “percent sequence identity” between two polypeptide orpolynucleotide sequences refers to the number of identical matchedpositions shared by the sequences over a comparison window, taking intoaccount additions or deletions (i.e., gaps) that must be introduced foroptimal alignment of the two sequences. A matched position is anyposition where an identical nucleotide or amino acid is presented inboth the target and reference sequence. Gaps presented in the targetsequence are not counted since gaps are not nucleotides or amino acids.Likewise, gaps presented in the reference sequence are not counted sincetarget sequence nucleotides or amino acids are counted, not nucleotidesor amino acids from the reference sequence.

The percentage of sequence identity is calculated by determining thenumber of positions at which the identical amino-acid residue or nucleicacid base occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. The comparison ofsequences and determination of percent sequence identity between twosequences can be accomplished using readily available software both foronline use and for download. Suitable software programs are availablefrom various sources, and for alignment of both protein and nucleotidesequences. One suitable program to determine percent sequence identityis bl2seq, part of the BLAST suite of program available from the U.S.government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq performs a comparison between twosequences using either the BLASTN or BLASTP algorithm. BLASTN is used tocompare nucleic acid sequences, while BLASTP is used to compare aminoacid sequences. Other suitable programs are, e.g., Needle, Stretcher,Water, or Matcher, part of the EMBOSS suite of bioinformatics programsand also available from the European Bioinformatics Institute (EBI) atwww.ebi.ac.uk/Tools/psa.

Different regions within a single polynucleotide or polypeptide targetsequence that align with a polynucleotide or polypeptide referencesequence can each have their own percent sequence identity. It is notedthat the percent sequence identity value is rounded to the nearesttenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to80.2. It also is noted that the length value will always be an integer.

In certain aspects, the percentage identity “X” of a first amino acidsequence to a second sequence amino acid is calculated as 100×(Y/Z),where Y is the number of amino acid residues scored as identical matchesin the alignment of the first and second sequences (as aligned by visualinspection or a particular sequence alignment program) and Z is thetotal number of residues in the second sequence. If the length of afirst sequence is longer than the second sequence, the percent identityof the first sequence to the second sequence will be higher than thepercent identity of the second sequence to the first sequence.

One skilled in the art will appreciate that the generation of a sequencealignment for the calculation of a percent sequence identity is notlimited to binary sequence-sequence comparisons exclusively driven byprimary sequence data. Sequence alignments can be derived from multiplesequence alignments. One suitable program to generate multiple sequencealignments is ClustalW2, available from www.clustal.org. Anothersuitable program is MUSCLE, available from www.drive5.com/muscle/.ClustalW2 and MUSCLE are alternatively available, e.g., from the EBI.

It will also be appreciated that sequence alignments can be generated byintegrating sequence data with data from heterogeneous sources such asstructural data (e.g., crystallographic protein structures), functionaldata (e.g., location of mutations), or phylogenetic data. A suitableprogram that integrates heterogeneous data to generate a multiplesequence alignment is T-Coffee, available at www.tcoffee.org, andalternatively available, e.g., from the EBI. It will also be appreciatedthat the final alignment used to calculate percent sequence identity canbe curated either automatically or manually.

As used herein, the term “antibody” refers to at least the minimalportion of an antibody which is capable of binding to antigen, e.g.IL23, comprising at least the variable domain of a heavy chain (VH) andthe variable domain of a light chain (VL) in the context of a typicalantibody produced by a B cell. Basic antibody structures in vertebratesystems are relatively well understood. See, e.g., Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988).

Antibodies or antigen-binding fragments, variants, or derivativesthereof include, but are not limited to, polyclonal, monoclonal, human,humanized, or chimeric antibodies, single chain antibodies,epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv), fragments comprising either a VL or VH domain, fragmentsproduced by a Fab expression library. ScFv molecules are known in theart and are described, e.g., in U.S. Pat. No. 5,892,019. Immunoglobulinor antibody molecules encompassed by this disclosure can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Thus, in view if this definition of the term “antibody,” references toan antibody, e.g., the anti-IL23 antibody MEDI2070, refer to theMEDI2070 antibody and also to antigen-binding fragments, variants, andderivatives thereof. In some aspects, the antibody is an anti-IL23antibody which can specifically bind to the p19 subunit of IL23 (SEQ IDNO: 15), to the p40 subunit of IL23 (SEQ ID NO: 16), or both.

In some aspects, the anti-IL23 antibody or antigen-binding fragmentthereof comprises, consists, or consists essentially of the heavy chain(HC) (SEQ ID NO: 17) and/or the light chain (SEQ ID NO:18) of MEDI2070,or an antigen-binding fragment, variant, or derivative thereof. In someaspects, the anti-IL23 antibody or antigen-binding fragment thereofcomprises, consists, or consists essentially of the heavy chain variableregion (VH) (SEQ ID NO: 7) and/or the light chain variable region (VL)(SEQ ID NO: 8) of MEDI2070. In some aspects, the anti-IL23 antibody orantigen-binding fragment thereof comprises at least one of thecomplementarity determining regions of MEDI2070 (SEQ ID NOS: 33-38),e.g., it comprises one, two, three, four, five, or the sixcomplementarity determining regions of MEDI2070.

In some aspects, the anti-IL23 antibody or antigen-binding fragmentthereof comprises a VH region comprising, consisting, or consistingessentially of the sequence of SEQ ID NO: 45 and/or a VL regioncomprising, consisting, or consisting essentially of the sequence of SEQID NO: 46, or an antigen-binding fragment, variant, or derivativethereof. In some aspects, the anti-IL23 antibody or antigen-bindingfragment thereof comprises at least one of the complementaritydetermining regions of SEQ ID NOS: 47-49 (CDRs of the VH of SEQ IDNO:45) and/or SEQ ID NOS: 50-52 (CDRs of the VL of SEQ ID NO: 46). SeeU.S. Pat. No. 8,722,033, which is herein incorporated by reference inits entirety.

In other aspects, the IL23 antagonist is an anti-IL23 antibody selectedfrom ustekinumab or briakinumab (targeting the p40 subunit of IL23),guselkumab, tildrakizumab, BI-655066 or LY-3074828 (targeting the p19subunit of IL23), an antigen-binding fragment thereof, or a combinationthereof. In other aspects, the IL23 antagonist is a molecule competingwith ustekinumab or briakinumab (targeting the p40 subunit of IL23),guselkumab, tildrakizumab, BI-655066 or LY-3074828 (targeting the p19subunit of IL23), an antigen-binding fragment thereof, or a combinationthereof, for binding to IL23.

In some aspects, the antibody is a naturally-occurring antibody, e.g.,an antibody isolated and/or purified from a mammal, or produced by ahybridoma generated from a mammalian cell. In some aspects, the antibodyis a genetically-engineered antibody, e.g., a humanized antibody, achimeric antibody, a CDR-grafted antibody, a humaneered antibody, abispecific antibody, a trispecific antibody, and the like. Geneticengineering techniques also provide the ability to make fully humanantibodies in a non-human source. In some aspects, the antibody isgenerated using phage display.

In some aspects, the antibody is in polymeric, oligomeric, or multimericform. In certain aspects in which the antibody comprises two or moredistinct antigen binding regions fragments, the antibody is consideredbispecific, trispecific, or multi-specific, or bivalent, trivalent, ormultivalent, depending on the number of distinct epitopes that arerecognized and bound by the antibody.

In some aspects, the binding agent is an antigen binding fragment of anyantibody of the preceding paragraphs. The antigen binding fragment (alsoreferred to herein as “antigen binding portion”) may be an antigenbinding fragment of any of the antibodies described herein. The antigenbinding fragment can be any part of an antibody that has at least oneantigen binding site, including, but not limited to, Fab, Fab′, F(ab′)2,Fd, Fvs, dsFv, disulfide-linked Fvs (sdFv), single chain Fvs (scFVs),fragments comprising either a light or heavy chain variable domain,single-chain antibodies, diabodies, triabodies, bis-scFvs, fragmentsexpressed by a Fab expression library, domain antibodies, VhH domains,V-NAR domains, VH domains, VL domains, and the like.

A typical antibody comprises at least two heavy (H) chains and two light(L) chains interconnected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as VH, VHregion, or VH domain) and a heavy chain constant region. The heavy chainconstant region is comprised of three or four constant domains, CH1,CH2, CH3, and CH4. Each light chain is comprised of a light chainvariable region (abbreviated herein as VL, VL region, or VL domain) anda light chain constant region. The light chain constant region iscomprised of one domain, CL.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed Complementarity Determining Regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FW). Each VH and VL is composed of three CDRs and four FWs,arranged from amino-terminus to carboxy-terminus in the following order:FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. Framework regions can bedesignated according to their respective VH and VL regions. Thus, e.g.,VH-FW1 would refer to the first framework region of VH. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies canmediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (C1q) of the classical complement system.

By “specifically binds,” it is generally meant that an antibody (e.g.,an anti-IL23 antibody) or fragment, variant, or derivative thereof bindsto an epitope via its antigen-binding domain, and that the bindingentails some complementarity between the antigen binding domain and theepitope. According to this definition, an antibody is said to“specifically bind” to an epitope when it binds to that epitope via itsantigen-binding domain more readily than it would bind to a random,unrelated epitope.

The term “epitope” as used herein refers to an antigenic proteindeterminant capable of binding to an antibody (e.g., an anti-IL23antibody) or fragment, variant, or derivative thereof binds disclosedherein. In some aspects, the term epitope refers to a proteindeterminant (e.g., an amino acid sequence) of the p19 subunit of IL23.In some aspects, the term epitope refers to a protein determinant (e.g.,an amino acid sequence) of the p40 subunit of IL23. Epitopes usuallyconsist of chemically active surface groupings of molecules such asamino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. The part of an antibody or binding molecule thatrecognizes the epitope is called a paratope. The epitopes of proteinantigens are divided into two categories, conformational epitopes andlinear epitopes, based on their structure and interaction with theparatope. A conformational epitope is composed of discontinuous sectionsof the antigen's amino acid sequence. These epitopes interact with theparatope based on the 3-D surface features and shape or tertiarystructure of the antigen. By contrast, linear epitopes interact with theparatope based on their primary structure. A linear epitope is formed bya continuous sequence of amino acids from the antigen.

The term “antibody binding site” refers to a region in the antigen(e.g., an amino acid sequence of the p19 subunit of IL23, or an aminoacid sequence of the p40 subunit of IL23) comprising a continuous ordiscontinuous site (i.e., an epitope) to which a complementary antibodyspecifically binds. Thus, the antibody binding site can containadditional areas in the antigen which are beyond the epitope and whichcan determine properties such as binding affinity and/or stability, oraffect properties such as antigen enzymatic activity or dimerization.Accordingly, even if two antibodies bind to the same epitope within anantigen, if the antibody molecules establish distinct intermolecularcontacts with amino acids outside of the epitope, such antibodies areconsidered to bind to distinct antibody binding sites.

An antibody or fragment, variant, or derivative thereof is said tocompetitively inhibit binding of a reference antibody or antigen bindingfragment to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody or antigen binding fragment to the epitope. Competitiveinhibition can be determined by any method known in the art, forexample, competition ELISA assays. A binding molecule can be said tocompetitively inhibit binding of the reference antibody orantigen-binding fragment to a given epitope by at least 90%, at least85%, at least 80%, at least 75%, at least 70%, at least 65%, at least60%, at least 55%, at least 50%, at least 45%, at least 40%, at least35%, at least 30%, or at least 25%.

Antibodies or antigen-binding fragments, variants, or derivativesthereof disclosed herein can be described or specified in terms of theepitope(s) or portion(s) of an antigen, e.g., a target polysaccharidethat they recognize or specifically bind. For example, the portion ofIL23 that specifically interacts with the antigen-binding domain of ananti-IL23 antibody (e.g., MEDI2070) is an “epitope.”

The term “IL23-mediated disease or disorder,” used interchangeably with“IL23-mediated disease” herein, as well as grammatical variants thereof,refers to any pathology known in the art to be caused by (alone or inassociation with other mediators), exacerbated by, associated with, orprolonged by abnormal levels of IL23 or abnormal activation of the IL23pathway in the subject having the disorder. Non-limiting examplesinclude IL23-mediated inflammatory bowel diseases (e.g., Crohn'sdisease), as well as pulmonary diseases, chronic inflammatory skindiseases, inflammatory diseases, autoimmune diseases, neurodegenerativediseases, or cancer.

In some aspects, the IL23-mediated inflammatory bowel disease is CD,ulcerative colitis (UC), Behçet's disease, or celiac disease. In someaspects, the IL23-mediated pulmonary disease is asthma (e.g., allergicasthma, atopic asthma, corticosteroid naive asthma, chronic asthma,corticosteroid resistant asthma, corticosteroid refractory asthma,asthma due to smoking, or asthma uncontrolled on corticosteroids),idiopathic pulmonary fibrosis (IPF), or chronic obstructive pulmonarydisease (COPD).

In some aspects, the IL23-mediated chronic inflammatory skin disease isatopic dermatitis, allergic contact dermatitis, eczema, psoriasis,alopecia areata, or palmoplantar pustulosis. In some aspects, theIL23-mediated inflammatory disease is psoriatic arthritis, anklyosingspondylitis, arthritis, rheumatoid arthritis (RA), a rheumatic disorder,ANCA vasculitis, Bechet's disease, or autoimmune thyroiditis. In someaspects, the IL23-mediated autoimmune disease is multiple sclerosis(MS), Sjogren's syndrome (SS), systemic lupus erythematosus (SLE),autoimmune encephalomyelitis, collagen-induced arthritis, or type 1diabetes mellitus.

In some aspects, the IL23-mediated neurodegenerative disease isAlzheimer's disease. In some aspects, the IL23-mediated cancer ismelanoma, colorectal cancer, stomach cancer, myeloma, prostate cancer,colitis-associated cancer, ovarian cancer, oral cancer, esophagealcancer, leukemia hepatitis B virus (HBV)-related hepatocellularcarcinoma, breast cancer, lung cancer, and nasopharyngeal cancer. Insome aspects, the IL23-mediated disease or disorder is a microbialinfection, including, e.g., mycobacterial disease, or leishmaniasis. Insome aspects, the IL23-mediated disease or disorder is a fungal or aviral infection (see, e.g., Khader et al., Mucosal Immunol 2(5): 403-411(2009)).

As used herein, the terms “treat,” “treating,” “treatment,” or“treatment of” as used herein refers methods that are aimed at (1) toreducing the potential for an IL23-mediated disease or disorder, e.g.,delaying or preventing the onset of an IL23-mediated disease ordisorder, (2) reducing the occurrence of the IL23-mediated disease ordisorder, e.g., slowing down or stopping the progression or aggravationof the symptoms or physical deterioration associated with theIL23-mediated disease or disorder, (3) a reduction in the severity oramelioration of the symptoms of the IL23-mediated disease or disorder,preferably, to an extent that the subject no longer suffers discomfortand/or altered function due to it (for example, a relative reduction inCD exacerbations or disease severity when compared to untreatedpatients), and/or (4) curing the IL23-mediated disease or disorder.

Unless otherwise specified, the terms “treat,” “treating,” “treatment,”or “treatment of” (or grammatically equivalent terms) refer to bothprophylactic and therapeutic treatment regimes. Thus, a treatment may beadministered prior to the onset of the disease, for a prophylactic orpreventive action. It may also be administered after initiation of theIL23-mediated disease or disorder, for a therapeutic action. In someaspects, treatment of an IL23-mediated disease or disorder can comprisesurgery.

The terms “subject” or “patient” as used herein refer to any subject,particularly a mammalian subject, for whom diagnosis, prognosis, ortherapy of an IL23-mediated disease or disorder is desired. As usedherein, the terms “subject” or “patient” include any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows, bears, chickens, amphibians, reptiles, etc. As usedherein, phrases such as “a patient having an IL23-mediated disease”includes subjects, such as mammalian subjects, including humans thatwould benefit from the administration of a therapy, imaging or otherdiagnostic procedure, and/or preventive treatment for that IL23-mediateddisease.

In some aspects, a subject is a naïve subject. A “naïve subject” is asubject that has not been administered a therapy, for example atherapeutic agent. In some aspects, a naïve subject has not been treatedwith a therapeutic agent prior to being diagnosed as having anIL23-mediated disease. In some aspects, a subject has received therapyand/or one or more doses of a therapeutic agent (e.g., a therapeuticagent capable of modulating an IL23-mediated disease) prior to beingdiagnosed as having an IL23-mediated disease.

In some aspects, the term “patient having an IL23-mediated disease ordisorder,” for example a patient having a specific IL23-mediated diseasesuch as Crohn's disease (i.e., a “Crohn's disease patient” or “CDpatient”), refers to a subject that presents one or more symptomsindicative of the IL23-mediated disease or disorder (e.g., CD) or thatis screened for a particular IL23-mediated disease or disorder (e.g.,CD). Alternatively or additionally, the term “patient having anIL23-mediated disease” also encompasses subjects suspected of having anIL23-mediated disease or disorder (e.g., CD) or who may have one or morerisk factor (e.g., age, sex, family history, etc). The term alsoencompasses subjects that have not been tested for an IL23-mediateddisease or disorder, as well as subjects that have received an initialdiagnosis.

The term “therapy” as used herein includes any means for curing,mitigating, or preventing an IL23-mediated disease or disorder,including, for example, therapeutic agents, instrumentation, supportivemeasures, and surgical or rehabilitative procedures. In this respect,the term therapy encompasses any protocol, method and/or therapeutic ordiagnostic that can be used in prevention, management, treatment, and/oramelioration of an IL23-mediated disease or disorder.

The term “therapeutic agent” as used herein refers to anytherapeutically active substance that is administered to a subjecthaving an IL23-mediated disease or disorder to produce a desired,usually beneficial, effect. The term therapeutic agent includes, e.g.,classical low molecular weight therapeutic agents commonly referred toas small molecule drugs and biologics including but not limited toantibodies or active fragments thereof, peptides, protein drugs, proteinconjugate drugs, etc. A therapeutic agent can also be a pro-drug, whichmetabolizes into the desired therapeutically active substance whenadministered to a subject. In some aspects, the therapeutic agent is aprophylactic agent. In addition, a therapeutic agent can bepharmaceutically formulated.

As used herein, the term “effective amount” or “pharmaceuticallyeffective amount” or “therapeutically effective amount” refers to aquantity of the compound(s) in a preparation which, when administered toa subject is sufficient to achieve a desired therapeutic and/orprophylactic effect, e.g., an amount which alleviates a symptom,ameliorates a condition, or slows the onset of disease conditionsaccording to clinically acceptable standards for the disorder orcondition to be treated. The amount of a composition administered to thesubject will depend on the type and severity of the IL23-mediateddisease or disorder and on the characteristics of the individual, suchas general health, age, sex, body weight and tolerance to drugs. It willalso depend on the degree, severity and type of disease. The skilledartisan will be able to determine appropriate dosages depending on theseand other factors. The compositions can also be administered incombination with one or more additional therapeutic compounds and/ortreatments.

The term “biological sample” is used herein in its broadest sense. Themethods disclosed herein can be carried out using any sample that maycontain the disclosed biomarkers, i.e., CCL20 alone or in combinationwith at least one additional biomarker, e.g., IL22, LCN2, CRP, FCP, or acombination thereof. A biological sample is generally obtained from asubject. A sample may be of any biological tissue or fluid with whichbiomarkers of the present disclosure may be assayed. Frequently, asample will be a “clinical sample”, i.e., a sample derived from apatient.

With regard to the methods disclosed herein, non-limiting examples ofthe sample obtained from the subject comprises, for example, wholeblood, blood serum, plasma, saliva, sputum, bronchoalveolar lavagefluid, cerebrospinal fluid, pleural fluid, pericardial fluid, ascites,synovial fluid, epithelial cells, urine, stool, skin, tissue, pinch orfine needle biopsy samples, and archival samples with known diagnosis,treatment and/or outcome history. Biological samples may also includesections of tissues such as frozen sections taken for histologicalpurposes, or combinations thereof. Samples can be obtained by any meansknown in the art.

The term “biological sample” also encompasses any material derived byprocessing a biological sample. Derived materials include, but are notlimited to, proteins extracted from the sample, nucleic acids extractfrom the sample, or nucleic acid sequences PCR amplified from thesample. Processing of a biological sample may involve one or more of:filtration, distillation, extraction, concentration, inactivation ofinterfering components, addition of reagents, and the like. For example,a blood sample can be fractionated into serum or into fractionscontaining particular types of cells

In some aspects, a sample can be a combination of samples from anindividual, such as a combination of a tissue and fluid sample. Thesample can be pretreated as necessary by dilution in an appropriatebuffer solution or concentrated. Any of a number of standard aqueousbuffer solutions and/or protease inhibitor, employing any of a varietyof buffers, such as phosphate, TRIS, or the like, at physiological pH,can be used. In some aspects, a sample can be a combination of samplesfrom multiple individuals.

The samples obtained to diagnose and/or determine expression levels ofbiomarkers for a certain IL23-mediated disease or disorder can varydepending on the specific methods used in the art. Thus, samples can be,for example, biological samples (whole blood, blood serum, plasma,saliva, sputum, bronchoalveolar lavage fluid, stool, epithelial cells,tissue biopsy samples, intestinal tissue biopsy samples, urine, skin,polyps, cerebrospinal fluid, pleural fluid, pericardial fluid, ascites,synovial fluid. etc), physical exam data (e.g., presence of fever, painand/or abdominal tenderness), family history, results from laboratorytests (e.g., blood protein levels, blood sedimentation rates, bodymineral levels, red blood cell counts, white blood cell counts, presenceof blood and/or infectious microbes in stool samples), imaging studiesand endoscopy (e.g., barium X-rays and other X-rays, CT scans,colonoscopy, sigmoidoscopy, video capsule endoscopy), etc.

As used herein, the term “control”, when used to characterize a subject,refers to a subject that is healthy or to a patient who has beendiagnosed with a specific disease other than an IL23-mediated disease ordisorder. The term “control sample” refers to one, or more than one,biological samples obtained from a healthy subject or from a patientdiagnosed with a disease other than an IL23-mediated disease ordisorder.

A sample from a patient can be obtained before or after theadministration of a therapy to treat an IL23-mediated disease ordisorder. In some cases, successive samples can be obtained from thepatient after therapy has commenced or after therapy has ceased. Samplescan, for example, be requested by a healthcare provider (e.g., a doctor)or healthcare benefits provider, obtained and/or processed by the sameor a different healthcare provider (e.g., a nurse, a hospital) or aclinical laboratory, and after processing, the results can be forwardedto the original healthcare provider or yet another healthcare provider,healthcare benefits provider or the patient.

Similarly, the quantification of the expression level of a biomarkerdisclosed herein, i.e., CCL20 alone or in combination with at least oneadditional biomarker, e.g., IL22, LCN2, CRP, FCP, or a combinationthereof; comparisons and/or ratios between biomarker gene or proteinexpression levels; evaluation of the absence or presence biomarkers;determination of biomarker levels with respect to a certain threshold;treatment decisions; or combinations thereof, can be performed by one ormore healthcare providers, healthcare benefits providers, and/orclinical laboratories.

As used herein, the term “healthcare provider” refers to individuals orinstitutions that directly interact and administer to living subjects,e.g., human patients. Non-limiting examples of healthcare providersinclude doctors, nurses, technicians, therapist, pharmacists,counselors, alternative medicine practitioners, medical facilities,doctor's offices, hospitals, emergency rooms, clinics, urgent carecenters, alternative medicine clinics/facilities, and any other entityproviding general and/or specialized treatment, assessment, maintenance,therapy, medication, and/or advice relating to all, or any portion of, apatient's state of health, including but not limited to general medical,specialized medical, surgical, and/or any other type of treatment,assessment, maintenance, therapy, medication, and/or advice.

As used herein, the term “clinical laboratory” refers to a facility forthe examination or processing of materials derived from a livingsubject, e.g., a human being. Non-limiting examples of processinginclude biological, biochemical, serological, chemical,immunohematological, hematological, biophysical, cytological,pathological, genetic, or other examination of materials derived fromthe human body for the purpose of providing information, e.g., for thediagnosis, prevention, or treatment of any disease or impairment of, orthe assessment of the health of living subjects, e.g., human beings.These examinations can also include procedures to collect or otherwiseobtain a sample, prepare, determine, measure, or otherwise describe thepresence or absence of various substances in the body of a livingsubject, e.g., a human being, or a sample obtained from the body of aliving subject, e.g., a human being.

A clinical laboratory can, for example, collect or obtain a sample,process a sample, submit a sample, receive a sample, transfer a sample,analyze or measure a sample, quantify a sample, provide the resultsobtained after analyzing/measuring/quantifying a sample, receive theresults obtained after analyzing/measuring/quantifying a sample,compare/score the results obtained after analyzing/measuring/quantifyingone or more samples, provide the comparison/score from one or moresamples, obtain the comparison/score from one or more samples, or otherrelated activities.

As used herein, the term “healthcare benefits provider” encompassesindividual parties, organizations, or groups providing, presenting,offering, paying for in whole or in part, or being otherwise associatedwith giving a patient access to one or more healthcare benefits, benefitplans, health insurance, and/or healthcare expense account programs.

In some aspects, a healthcare provider can administer or instructanother healthcare provider to administer a therapy to treat anIL23-mediated disease or disorder. A healthcare provider can implementor instruct another healthcare provider or patient to perform, forexample, the following actions: obtain a sample, process a sample,submit a sample, receive a sample, transfer a sample, analyze or measurea sample, quantify a sample, provide the results obtained afteranalyzing/measuring/quantifying a sample, receive the results obtainedafter analyzing/measuring/quantifying a sample, compare/score theresults obtained after analyzing/measuring/quantifying one or moresamples, provide the comparison/score from one or more samples, obtainthe comparison/score from one or more samples, administer a therapy(e.g., a therapeutic agent that treats a IL23-mediated disease ordisorder), commence the administration of a therapy, cease theadministration of a therapy, continue the administration of a therapy,temporarily interrupt the administration of a therapy, increase theamount of an administered therapeutic agent, decrease the amount of anadministered therapeutic agent, continue the administration of an amountof a therapeutic agent, increase the frequency of administration of atherapeutic agent, decrease the frequency of administration of atherapeutic agent, maintain the same dosing frequency on a therapeuticagent, replace a therapy or therapeutic agent by at least anothertherapy or therapeutic agent, combine a therapy or therapeutic agentwith at least another therapy or additional therapeutic agent.

In some aspects, a healthcare benefits provider can authorize or deny,for example, collection of a sample, processing of a sample, submissionof a sample, receipt of a sample, transfer of a sample, analysis ormeasurement a sample, quantification a sample, provision of resultsobtained after analyzing/measuring/quantifying a sample, transfer ofresults obtained after analyzing/measuring/quantifying a sample,comparison/scoring of results obtained afteranalyzing/measuring/quantifying one or more samples, transfer of thecomparison/score from one or more samples, administration of a therapyor therapeutic agent, commencement of the administration of a therapy ortherapeutic agent, cessation of the administration of a therapy ortherapeutic agent, continuation of the administration of a therapy ortherapeutic agent, temporary interruption of the administration of atherapy or therapeutic agent, increase of the amount of administeredtherapeutic agent, decrease of the amount of administered therapeuticagent, continuation of the administration of an amount of a therapeuticagent, increase in the frequency of administration of a therapeuticagent, decrease in the frequency of administration of a therapeuticagent, maintain the same dosing frequency on a therapeutic agent,replace a therapy or therapeutic agent by at least another therapy ortherapeutic agent, or combine a therapy or therapeutic agent with atleast another therapy or additional therapeutic agent.

In addition, a healthcare benefits provider can, e.g., authorize or denythe prescription of a therapy, authorize or deny coverage for therapy,authorize or deny reimbursement for the cost of therapy, determine ordeny eligibility for therapy, etc.

II. CCL20 as a Clinical Response Biomarker

The recent discovery of a new CD4⁺ T cell subset, Th17, has transformedour understanding of the pathogenic basis of an increasing number ofchronic immune-mediated diseases. Particularly in tissues that interfacewith the microbial environment—such as the intestinal and respiratorytracts and the skin—where most of the Th17 cells in the body reside,dysregulated immunity to self (or the extended self, the diversemicrobiota that normally colonize these tissues) can result in chronicinflammatory disease. Various inflammatory conditions have potentialtargets for pharmacological development along the IL23 pathway. Theseinclude, for example, IL23, which affects the differentiation of Th17cells.

Some IL23-mediated diseases and disorders, for example CD or psoriasis,are heterogeneous diseases showing a wide spectrum of symptoms, severityand therefore drug responsiveness. One of the problems in clinicalmanagement of IL23-mediated diseases and disorders is the difficulty toidentify specific subgroups of patients undergoing early clinicalflare-up of symptoms for a timely and appropriate treatment, or theidentification of specific subgroups of patients that may or may notbenefit from a certain therapy, e.g., treatment with an IL23-antagonist.Thus, there still remains a need in the art for methods of identifyingand treating specific groups of patients having an IL23-mediated diseasewho are responsive to specific anti-IL23 therapies.

It has been reported that several genes and proteins are overexpressedin the serum and other tissues of patients with IL23-mediated diseasesand disorders, for example, CCL20. CCL20, like human beta-defensin(hBD)-2, is a potent chemoattractant for CCR6-positive immaturedendritic cells and T cells. CCL20 is increased in rheumatoid arthritisand psoriasis (Melis et al., Ann. Rheum. Dis. 69:618-23 (2010); Mrabetet al. Rheumatology International 33:265-66 (2013); Schlenk et al. J.Rheumatol. 32:2291-98 (2005); Homey et al. J. Immunol. 164:6621-32(2000); Lisignoli et al. J. Cell. Physiol. 210:798-806 (2007)), Crohn'sdisease and ulcerative colitis (Kaser et al. J. Clin. Immunol. 24:74-85(2004); Uiterweer et al. J. Clin. Immunol. 24:74-85 (2004); Lee et al.Inflammatory Bowel Diseases 11:1070-79 (2005); Uchida et al.Gastroenterology Research and Practice 2015:856532 (2015)), multiplesclerosis (Ambrosini et al., J. Neuropathol. Exp. Neurol. 64:706-15(2005)), Pseudomonas aeruginosa infections (Lin et al., Infect. Immun.71:365-73 (2003)), atopic dermatitis (Nakayama et al. Int. Immunol.13:95-103 (2001)), melanoma (Hasan et al. J. Immunol. 176:6512-22(2006)), periodontal disease and tooth decay (Veerayutthwilai et al.Oral Microbiol. Immunol. 22:5-13 (2007); Hosokawa et al., Clin. Exper.Immunol. 142: 285-91 (2005)), colorectal cancer (Rubie et al. TumourBiol. 27:166-74 (2006); Frick et al. Scand. J. Immunol. 78:298-305(2013)), dry eye disease (Dohlman et al. Invest. Ophthalmol. Vis. Sci.54:4081-91 (2013)), ulcerative colitis-associated neoplasia (Hashimotoet al., Oncology Letters 6:1271-1276 (2013), spontaneous intestinaltumorigenesis (Nandi et al. PLoS One 9:e97566 (2014)), COPD (Brand etal. J. Biol. Chem. 290:14717-28 (2015)), etc. See also Schutyser et al.,Cytokine and Growth Factor Reviews 14: 409-426 (2003). U.S. Publ. No.2011/0212104 disclosed that levels of a number of molecules, includingCCL20 were elevated in CD patients and proposed CCL20 as a potentialbiomarker for IBD (including CD and UC). None of the references citedabove disclosed thresholds, levels or ranges of CCL20 that could be usedto stratify or identify a population of patients having an IL23-mediateddisease and/or to select subpopulations of patients having anIL23-mediated disease for treatment with a specific IL23 antagonist(e.g., an anti-IL23 antibody targeting, e.g., the p19 subunit of IL23).Further none of the cited references discloses an inverse correlationbetween decreasing CCL20 levels in a population of patients having anIL23-mediated disease and increased response to anti-IL23 therapies.More importantly with respect to the present disclosure none of thecited references suggest that low or decreased levels of CCL20 could beused to stratify or identify a population of patients having anIL23-mediated disease for treatment with a specific IL23 antagonist(e.g., an anti-IL-23 antibody targeting, e.g., the p19 subunit of IL23).

New therapeutic options, e.g., antibodies specifically targeting IL23such as MEDI2070, have the potential to address the unmet medical needsof patients suffering from IL23-mediated diseases. Accordingly, means toidentify, for example, groups of patients who are likely to have a goodclinical response to MEDI2070 (e.g., a diagnostic biomarker orcombination thereof) and/or other anti-IL23 therapeutics known in theart, could greatly augment their utility.

In general, the methods disclosed herein are based on

-   (a) the detection of changes in the levels of CCL20 alone, or in    combination with the detection of changes in the levels of one, two,    three, or more biomarkers, including, e.g., IL22, LCN2, CRP, and/or    FCP, in patients with IL23-mediated diseases or disorders (e.g.,    CD), and-   (b) the correlation of these changes with increased clinical    response to therapy with an IL23 antagonist (including, for example,    an anti-IL23 antibody or antigen binding fragment thereof targeting,    e.g., the p19 subunit of IL23).

In other words, specific levels of CCL20 alone, or in combination withIL22 and/or,

LCN2, and further in combination with other biomarkers such as CRPand/or FCP, or any combination thereof, are correlated with clinicalefficacy of therapies and useful to predict clinical outcomes inspecific populations of patients suffering from an IL23-mediated diseaseor disorder, e.g., CD.

The term “IL23 pathway biomarker” as used herein encompasses proteins inthe IL23 pathway including chemokine (CC motif) ligand 20 (CCL20,MIP-3a) and other IL23 pathway proteins such as interleukin-22 (IL22),lipocalin-2 (LCN2), interleukin-17F (IL17F), IL17A/IL17F (IL17A/F), S100calcium-binding protein A12 (S100A12), interleukin 23 receptor (IL23R),interleukin 12B (IL12B), interleukin 23A (IL23A), defensin B2 (DEFB-2,DEFB-4), IL1β, serine (or cysteine) proteinase inhibitor member 3(SERPINB3), tumor necrosis factor alpha (TNF-α), CCL6, α3 integrin,interleukin-21 (IL21), CCR6, CCL22, IL1R1, IFNγ, PI3/Elafin, LL37, RORγ,RORγT, IL26, S100A7, DEFB103B, GM-CSF, or combinations thereof. See,e.g., Haider et al. J. Immunol. 180: 1913-1920 (2008); Nakae et al. J.Leukocyte Biol. 81: 1258-1268 (2007); Guttman-Yassky et al. J Immunol.181(10): 7420-7427 (2008), Wilson, et al. Nature Immunology 8:950-7(2007); all of which are herein incorporated by reference in theirentireties. In some aspects, the IL23 pathway biomarker or biomarkersused by the methods disclosed herein comprises CCL20, alone or incombination with the IL23 pathway biomarkers IL22 and/or LCN2. In someaspects, the IL23 pathway or set of IL23 pathway biomarkers disclosedherein can be combined with additional biomarkers such as CRP and/orFCP,

In some aspects, CCL20 can be combined with other IL23 pathwaybiomarkers such as IL22 and/or LCN2, which in turn can be substituted orcombined with one or more molecules present in other inflammationpathways known in the art, and/or with other biomarkers linked toIL23-mediated diseases or disorders known in the art, including, but notlimited to, for example, C-reactive protein (CRP), calprotectin(S100A8/S100A9 complex), DMBT1, MIF, PAP/REG3α, REG3γ, haptoglobin,interleukin-6 (IL6), lactoferrin, GP-39 (YKL-40), GPX-2, GPX-3,neutrophil elastase, etc.

The term “biomarker” refers to a factor that is a distinctive indicatorof a biological process, biological event, and/or pathologic condition,e.g., a predictor of clinical response to treatment with an IL23antagonist (e.g., an anti-IL23 antibody targeting, e.g., the p19 subunitof IL23, or an antigen-binding fragment thereof). As used herein, theterm biomarker encompasses both clinical markers and biological markers.Thus, in the context of the present invention, the term “biomarker”encompasses, e.g., “biological biomarkers” comprises, for example, CCL20(alone or in combination with other IL23 pathway biomarkers such as IL22and/or LCN2), other biomarkers linked to IL23-mediated diseases ordisorders (e.g., CRP and/or calprotectin), and combinations thereof. Thebiological markers disclosed herein also include the genes encodingthose proteins (DNA and/or RNA), as well as metabolic products.

The term “biomarker” also encompasses “clinical biomarkers” (alsoreferred to as “clinical status markers”) that can be predictive ofresponse to biological therapies, for example, gender, age, concomitantdrugs, smoking status, body mass index (BMI), etc. See, e.g., U.S. Publ.Nos. US20150065530, US20140141990, US20130005596, US20090233304, US20140199709, US20130303398, and US20110212104, which are hereinincorporated by reference in their entireties.

The biomarkers disclosed herein (e.g., CCL20, IL22 and/or LCN2) alsoinclude proteins having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to their respective wild type sequences (e.g., in the case ofCCL20, IL22, and LCN2 to SEQ ID NOS: 1, 4, and 6, respectively), andnucleic acids having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity totheir respective wild type sequence (e.g., in the case of CCL20, IL22,and LCN2 to SEQ ID NOS: 2, 3, and 5, respectively).

The biomarkers disclosed herein, e.g., CCL20, IL22, or LCN2, alsoinclude fragments, variants, and derivatives thereof. As used herein, a“variant” biomarker contains at least one amino acid sequence alterationas compared to the amino acid sequence of the corresponding wild-typepolypeptide. An amino acid sequence alteration can be, for example, asubstitution, a deletion, or an insertion of one or more amino acids,preferably conservative substitutions. A variant biomarker can have anycombination of amino acid substitutions, deletions or insertions. In oneaspect, a biomarker variant polypeptide can have an integer number ofamino acid alterations such that its amino acid sequence shares at least60, 70, 80, 85, 90, 95, 97, 98, 99, 99.5 or 100% identity with the aminoacid sequence of the corresponding wild-type polypeptide.

Chemokine Ligand 20 (CCL20)

The term “CCL20” as used herein refers to the chemokine ligand 20protein (NCBI Reference Sequence NP_004582.1; SEQ ID NO: 6) encoded bythe CCL20 gene (NCBI Reference Sequence NM_004591.2; SEQ ID NO: 5). Seealso Uniprot Acc. No. P7855 herein incorporated by reference in itsentirety. CCL20, also known as macrophage inflammatory protein 3 alpha(MP3a), CKb4, liver and activation-regulated chemokine (LARC), ST38,MIP3A, MIP-3a, small-inducible cytokine A20 (SSCYA20), or beta-chemokineexodus-1, is known to play an important role in dendritic celltrafficking and in the recruitment of activated T cells. It is producedby activated Th17 cells, and colonic epithelial cells are known to be amajor site of CCL20 production in irritable bowel disease.

The signal sequence for CCL20 is 26 amino acids long, i.e., residues27-96 of SEQ ID NO: 6 correspond to mature CCL20.

The term CCL20 also includes fragments (e.g., CCL20 can be cleaved intothree different chains corresponding to positions 27-93, 27-90, and28-96), isoforms (e.g., full length isoform 1, which is 96 amino acidslong; isoform 2, which is 95 amino acids long because it lacks thealanine at position 27, therefore resulting in a mature polypeptidelacking the N-terminal alanine residue of isoform 1), variants (e.g.,V47M, and other variants known in the art), and derivatives thereof(e.g., glycosylated or aglycosilated forms of the CCL20 protein, orotherwise chemically modified forms of the protein). In some aspects,the term CCL20 refers to the CCL20 gene, which includes genomic DNA,cDNA, mRNA, and fragments thereof. In some aspects, the term CCL20 alsorefers to oligonucleotides capable of specifically hybridizing to theCCL20 gene under stringent conditions.

Descriptions for other IL23 pathway biomarkers discussed in the instantapplication are provided below.

Interleukin 22 (IL22)

The term “IL22” as used herein refer to the interleukin 22 protein (NCBIReference Sequence NP_065386.1; SEQ ID NO: 4) encoded by the IL22 gene(NCBI Reference Sequence NM_020525.4; SEQ ID NO: 3). See also UniprotAcc. No. Q9GZX6 herein incorporated by reference in its entirety. IL22is a pro-inflammatory cytokine known to be expressed from Th17 cells.Wilson et al. Nature Immunol. 8:950 (2007). Increased serum levels ofIL22 have also been associated with IBD. See, e.g., Schmechel et al.Inflamm. Bowel Dis. 14:204 (2008); and Brand et al. Am. J. Physiol.Gastrointest. Liver Physiol 290:G827-G838 (2006). IL22 has been shown toinduce haptoglobin expression in hepatic cells (Dumoutier et al. Proc.Nat'l. Acad. Sci USA 97:10144 (2000)), to upregulate expression ofREG3α, REG3γ, S100A8, S100A9 and haptoglobin in colon epithelial cells(Zheng et al. Nature Medicine 14:282 (2008)), and to upregulateexpression of S100A8 and S100A9 in keratinocytes (Boniface et al. J.Immunol. 174:3695 (2005)).

IL22 has a 33 amino acid signal sequence, i.e., residues 34-179 of SEQID NO: 4 correspond to mature IL22. Xie et al. J. Biol. Chem. 275:31335(2000).

The term IL22 also includes fragments, variants (e.g., S158G and othervariants known in the art), and derivatives thereof (e.g., glycosylatedor aglycosilated forms of the IL22 protein, or otherwise chemicallymodified forms of the protein). In some aspects, the term IL22 refers tothe IL22 gene, which includes genomic DNA, cDNA, mRNA, and fragmentsthereof. In some aspects, the term IL22 also refers to oligonucleotidescapable of specifically hybridizing to the IL22 gene under stringentconditions.

Lipocalin-2 (LCN2)

The term “LCN2” as used herein refers to the lipocalin-2 protein (NCBIReference Sequence NP_005555.2; SEQ ID NO: 6) encoded by the LCN2 gene(NCBI Reference Sequence NM_005564.3; SEQ ID NO: 5). See also UniprotAcc. No. P80188 herein incorporated by reference in its entirety. LCN2,also known as 24p3 and neutrophil gelatinase-associated lipocalin (NGAL)(Kjeldsen et al. J. Biol. Chem. 268:10425-10432 (1993)), is a 25 kDasecretory glycoprotein that was originally identified in mouse kidneycells and human neutrophil granules. It belongs to the lipocalinsuperfamily that includes over 20 small secretory proteins, such asRBP4, fatty acid binding proteins (FABP), major urinary proteins (MUP),apolipoprotein D (apoD) and prostaglandin D synthases (PODS). SeeAkerstrom et al. Biochim Biophys Acta. 1482:1-8 (2000). The commonfeature of this protein family is their capacity to bind and transportsmall lipophilic substances, such as free fatty acids, retinoids,arachidonic acid and various steroids (Flower, Biochem J. 318:1-14(1996). LCN2 is expressed in colon epithelial cells and leukocytes inCrohn's disease colon.

LCN2 has a 20 amino acid signal sequence, i.e., residues 21-198 of SEQID NO: 6 correspond to mature LCN2. See Bundgaard et al. Biochem.Biophys. Res. Comm. 202:1468 (1994); International Appl. Publ. No. WO2006/091035; and U.S. Pat. App. Pub. No. 20050261191, which are hereinincorporated by reference in their entireties.

The term LCN2 also includes fragments, isoforms (e.g., isoform 2 inwhich the sequence DQCIDG (SEQ ID NO: 39) between residues 193 and 198is replaced with the sequence GNGQSG (SEQ ID NO: 40)), variants (e.g.,G9R, L13S, K82N, I155V, S178Y, and other variants known in the art), andderivatives thereof (e.g., glycosylated or aglycosilated forms of theLCN2 protein, or otherwise chemically modified forms of the protein). Insome aspects, the term LCN2 refers to the LCN2 gene, which includesgenomic DNA, cDNA, mRNA, and fragments thereof. In some aspects, theterm LCN2 also refers to oligonucleotides capable of specificallyhybridizing to the LCN2 gene under stringent conditions.

The methods disclosed herein can comprise the IL23 pathway biomarkersdisclosed above, for example, CCL20, IL22, or LCN2, and additionalbiomarkers such as CRP and/or FCP, which are described below.

C-Reactive Protein (CRP)

CCL20 and other IL23 pathway biomarkers disclosed herein, e.g., IL22and/or LCN2, can be combined for diagnostic, predictive, or monitoringpurposes with other IL23 pathway biomarkers, biomarkers downstream fromthe therapeutic intervention point (e.g., IL23), or biomarkers specificfor a certain IL23-mediated disease or disorders. For example, forinflammatory bowel disease (IBD) (including, e.g., CD or UC), suchspecific biomarkers include C-reactive protein (CRP) and/or fecalcalprotectin (FCP), which are described below.

The term “CRP” as used herein refers to the C-reactive protein (NCBIReference Sequence NP_000558.2; SEQ ID NO: 10) encoded by the CRP gene(NCBI Reference Sequence NM_000567.2; SEQ ID NO: 9). See also UniprotAcc. No. P02741 herein incorporated by reference in its entirety. CRP(also known as PTX1) has long been recognized as an acute phase reactantthat rises dramatically in concentration after tissue injury orinflammation. Elevated CRP levels have been associated with coronaryartery disease. CRP has long been used as a marker for inflammatorybowel disease, especially Crohn's disease. See, e.g., Vermerie et cd.(2004) Inflamm. Bowel Dis. 10:661 and Keshet et al. (2009) Am. J. Med.ScL 337:248. For use as a biomarker in humans, CRP levels may bedetermined using a custom-designed ELISA assay, or, for example, using acommercially available ELISA kit, such as the QUANTIKINE® human CRPImmunoassay kit from R&D Systems (Minneapolis, Minn., USA) and theAssayMax® human lactoferrin ELISA kit from AssayPro (St. Charles, Mo.,USA).

CRP has an 18 amino acid signal sequence, i.e., residues 19-224 of SEQID NO: 10 correspond to mature CRP.

The term CRP also includes fragments, variants (e.g., K1G, T1G, L170V,substitution of the sequence YSIFSYATKRQDNEIL (SEQ ID NO: 41) startingat position 16 with the sequence TVFSRMPPRDKTMRFF (SEQ ID NO: 42),deletion of sequence from position 80 to 98, and other variants known inthe art), and derivatives thereof (e.g., the pyrrolidine N-terminalcarboxylic acid modified form of CRP produced after cleavage of theresidues 1-19 signal peptide, and other chemically modified forms of theprotein). In isoform 2 of CRP the sequence from position 67 to 199 ismissing. In some aspects, the term CRP refers to the CRP gene, whichincludes genomic DNA, cDNA, mRNA, and fragments thereof. In someaspects, the term CRP also refers to oligonucleotides capable ofspecifically hybridizing to the CRP gene under stringent conditions.

Fecal Calprotectin (S100A8/S100A9) (FCP)

The term “FCP” as used herein refers to fecal calprotectin. FCP is acomplex of the mammalian proteins S100A8 and S100A9 produced byneutrophils, monocytes, and epithelial cells under inflammatoryconditions, Foell et al. J. Leukocyte Biol. 81:28 (2007). Calprotectincan inhibit the growth of Staphylococcus aureus in abscesses bychelation of nutrient Mn²⁺ and Zn^(2±). Corbin et al. Science 319:962(2008). Calprotectin has long been associated with intestinalinflammation, and has been proposed as a marker for IBD. See, e.g.,Lugering et al. Digestion 56:406 (1995); von Roon et al. Am. J.Gastroenterol. 102:803 (2007); Leach et al. Scand. J. Gastroenterol.42:1321(2007); Langhorst et al. Am. J. Gastroenterol. 103:162 (2008).Its measurement in feces has been shown to be useful in detecting activeIBD and predicting recurrence of disease. Angriman et al. ClinicaChimica Acta 381:63 (2007). In 2006, the U.S. Food & Drug Administration(FDA) approved the PHICAL® Fecal Calprotectin Immunoassay (GenovaDiagnostics, Inc., Asheville, N.C., USA) for use in diagnosing IBD. SeeU.S. Pat. Nos. 4,833,074; 5,455,160; and 6,225,072. The test involves anELISA assay with colorimetric readout. Other human calprotectindetection kits are also commercially available. See, e.g., U.S. Pat. No.6,225,072. Detection of fecal calprotectin at levels greater than 100μg/g of stool is diagnostic for IBD. von Roon et al. Am. J.Gastroenterol. 102:803 (2007). According to literature provided with thePHICAL® Test fecal calprotectin levels of over 50 μg/g are regarded as a“positive” result, based on median fecal calprotectin levels of over1700 μg/g (e.g. 200-20.000 μg/g) in IBD patients and 25 μg/g normalhealthy subjects.

S100A8 (S100 calcium-binding protein A8) (NCBI Reference SequenceNP_002955.2; SEQ ID NO: 12), also known as calgranulin A, cysticfibrosis antigen, myeloid-related protein 8, granulocyte L1 protein,calprotectin L1L subunit, CFAG, leukocyte L1 complex light chain,migration inhibitory factor-related protein 8, MRP-8, S100calcium-binding protein A8, and urinary stone protein band A, is encodedby the S100A8 gene (NCBI Reference Sequence NM_002964.3; SEQ ID NO: 11).See also Uniprot Acc. No. P05109 herein incorporated by reference in itsentirety.

S100A9 (S100 calcium-binding protein A9) (NCBI Reference SequenceNP_002956.1; SEQ ID NO: 14), also known as calgranulin B, cysticfibrosis antigen B, myeloid-related protein 14, calprotectin L1Hsubunit, leukocyte L1 complex heavy chain, migration inhibitoryfactor-related protein 14, and MRP-14, is encoded by the S100A9 gene(NCBI Reference Sequence NM_002965.3; SEQ ID NO: 13).

Neither S100A8 nor S100A9 have a classical signal sequence. Rammes etal. J. Bio. Chem. 272:9496 (1997). See also Uniprot Acc. No. P06702herein incorporated by reference in its entirety. The term calprotectinalso includes fragments, variants of the S100A8 and S100A9 proteins. Forexample, the initiator methionine of the S100A8 can be removed to yieldN-terminally processed S100A8 protein comprising amino acid residues 2to 93. Also, the cysteine amino acid at position 42 in the S100A8protein can be S-nitrosylated to yield 5-nitrosocysteine. In a variantof the S100A8 protein, the sequence from amino positions 80 to 93,VAAHKKSHEESHKE (SEQ ID NO: 43) is replaced with WQPTKKAMKKATKSS (SEQ IDNO: 44). Known variants of the S100A9 subunit of calprotectin includeC3A, E36Q, M63A, E78Q, M81A, M83A, S6H, K25F, H28L, and H20R. Theinitiator methionine of S100A9 can be removed to yield N-terminallyprocessed S100A9 protein comprising amino acid residues 2 to 114. Afterremoval of the N-terminal Met, the N-terminal Thr can be blocked. Thecysteine amino acid at position 3 in S100A9 can also be transientlyS-nitrosylated to yield S-nitrosocysteine. S-nitrosylation of Cys3 isimplicated in LDL(ox)-induced S-nitrosylation of GAPDH at Cys247 througha transnitrosylase mechanism involving a iNOS-S100A8/S100A9 complex. Thehistidine at position 105 (His105) in S100A9 can also be modified topros-methylhistidine. In addition, the threonine at position 113(Thr113) of S100A9 can be phosphorylated by MAPK14 to yieldphosphothreonine.

In some aspects, the terms calprotectin or FCP refer to the genesencoding S100A8 and/or S100A9, which includes genomic DNA, cDNA, mRNA,and fragments thereof. In some aspects, the terms FCP or calprotectinalso refer to oligonucleotides capable of specifically hybridizing tothe genes encoding S100A8 or S100A9 under stringent conditions.

In some aspects of the present disclosure, the methods disclosed hereincan be practiced using, for example, any of the sets of biomarkersdisclosed below:

-   (a) CCL20 alone-   (b) CCL20 in combination with at least one additional IL23 pathway    biomarker, e.g.:    -   a. CCL20, and IL22    -   b. CCL20, and LCN2    -   c. CCL20, IL22, and LCN2-   (c) CCL20 in combination with at least one additional biomarker    related to inflammation, e.g.:    -   a. CCL20, and FCP (S100A8 and/or S100A9)    -   b. CCL20, and CRP    -   c. CCL20, FCP (S100A8 and/or S100A9), and CRP-   (d) CCL20 in combination with at least one additional IL23 pathway    biomarker and at least one additional biomarker related to    inflammation, e.g.:    -   a. CCL20, IL22, and FCP (S100A8 and/or S100A9)    -   b. CCL20, IL22, and CRP    -   c. CCL20, LCN2, and FCP (S100A8 and/or S100A9)    -   d. CCL20, LCN2, and CRP    -   e. CCL20, IL22, LCN2, and FCP (S100A8 and/or S100A9)    -   f. CCL20, IL22, LCN2, and CRP    -   g. CCL20, IL22, FCP (S100A8 and/or S100A9), and CRP    -   h. CCL20, LCN2, FCP (S100A8 and/or S100A9), and CRP    -   i. CCL20, IL22, LCN2, FCP (S100A8 and/or S100A9), and CRP-   (e) The combinations of biomarkers disclosed above in combination    with at least one of biomarker selected from the group consisting of    IL17F, IL17A/F, IL23R, IL12B, IL6, IL21, TNF, CCR6, CCL22, IL1R1,    IFN-γ, S100A12, DEFB-2, DEFB-4, Ill, SERPINB3, PI3/Elafin, LL37,    RORγ, RORγT, IL26, S100A7, DEFB103B, and GM-CSF.

In some aspects of the present disclosure, the methods disclosed hereincan be applied in IL23-mediated diseases such as inflammatory boweldisease (IBD) (including, e.g., CD or UC) by using a set of biomarkerscomprising IL22 and/or LCN2 and optional additional biomarker such asCRP and/or calprotectin, as well as any combination or subset thereof.

Throughout any sections of the instant application, the recitation oftwo or more biomarkers ending in “and/or,” e.g., “CCL20, IL22, and/orLCN2” encompasses the individual components of the list as well ascombinations thereof considering that each component in the list islinked by the term “and/or.” Thus, “CCL20, IL22 and/or LCN2” isequivalent to “CCL20 and/or IL22 and/or LCN2” and encompasses CCL20alone, IL22 alone, LCN2 alone, CCL20+IL22, CCL20+LCN2, IL22+LNC2, andCCL20+IL22+LCN2.

III. Detection and Quantification of CCL20 and Other Biomarkers

CCL20 and other biomarkers disclosed herein, e.g., IL22, LCN2, CRPand/or FCP (either their expressed protein levels, or their respectivenucleic acid levels, such as mRNA levels) can be detected and quantifiedby any of a number of methods well known to those of skill in the art.These methods include analytic biochemical methods such aselectrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), hyperdiffusionchromatography, mass spectroscopy and the like, or various immunologicalmethods such as fluid or gel precipitin reactions, immunodiffusion(single or double), immunohistochemistry, affinity chromatography,immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linkedimmunosorbent assay (ELISAs), chemi-luminescence immunoassay (CLIA),immunofluorescent assays, Western blotting, and the like.

In some aspects, the method used to detect and/or quantify CCL20 or anyother biomarker disclosed herein, e.g., IL22, LCN2, CRP and/or FCP,comprises measuring the level, concentration, or amount of RNA, e.g.,mRNA, encoded by the gene or gene segments in the sample. Levels of RNA,e.g., mRNA, may be measured by any technique known in the art, includingbut not limited to northern blotting or quantitative PCR (qPCR),including methods such as reverse transcription qPCR, real time qPCR,and end-point qPCR. Alternatively, “tag based” technologies, such asSerial analysis of gene expression (SAGE) and RNA-Seq, may be carriedout to provide a relative measure of the cellular concentration ofdifferent mRNAs.

In some aspects, the method used to detect and/or quantify CCL20 andother biomarkers disclosed herein comprises measuring the level,concentration, or amount of the protein product encoded by the gene orgene segments in the sample. Suitable methods of determining expressionlevels of protein products are known in the art and include immunoassays(e.g., Western blotting, an enzyme-linked immunosorbent assay (ELISA), aradioimmunoassay (RIA), or a sandwich immunoassay or animmunohistochemical assay).

For a general review of immunoassays, see Methods in Cell Biology Volume37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York(1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, eds.(1991). See also, e.g., U.S. Patent Application Publication No.2007/0212723 A1, Shang et al., Circulation Research 101: 1146-1154(2007); and International Patent Application Publication Nos.WO/2012/094651 and WO/2010/129964.

In some aspects, the biomarkers disclosed herein, e.g., CCL20, IL22,LCN2, FCP and/or CRP, can be detected and/or quantified in anelectrophoretic polypeptide separation (e.g., a 1- or 2-dimensionalelectrophoresis). Means of detecting polypeptides using electrophoretictechniques are well known to those skilled in the art (see generally, R.Scopes (1982) Polypeptide Purification, Springer-Verlag, N.Y.;Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to PolypeptidePurification, Academic Press, Inc., N.Y.).

In some aspects, a Western blot (immunoblot) analysis is used to detectand quantify the presence of CCL20, IL22, LCN2, FCP, CRP, or anycombination thereof in the sample. This technique generally comprisesseparating sample polypeptides by gel electrophoresis on the basis ofmolecular weight, transferring the separated polypeptides to a suitablesolid support (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with antibodiesthat specifically bind the analyte. Antibodies that specifically bind tothe analyte may be directly labeled or alternatively may be detectedsubsequently using labeled antibodies (e.g., labeled sheep anti-mouseantibodies) that specifically bind to a domain of the primary antibody.

In some aspects, the biomarkers disclosed herein, e.g., CCL20, IL22,LCN2, FCP and/or CRP, can be detected and/or quantified in thebiological sample using an immunoassay. For a general review ofimmunoassays, see also Methods in Cell Biology Volume 37: Antibodies inCell Biology, Asai, ed. Academic Press, Inc. New York (1993); Basic andClinical Immunology 7th Edition, Stites & Terr, eds. (1991). In someaspects, the immunoassay can use one or more CCL20, IL22, LCN2, FCP orCRP antibodies or antigen binding fragments thereof which recognizehuman CCL20, IL22, LCN2, FCP or CRP, respectively.

In some aspects, the immunoassay comprises a sandwich immunoassay, e.g.,an enzyme-linked immunosorbent assay (ELISA) or a sandwichelectrochemiluminescent (ECL) assay, in which a first antibody orantigen-binding fragment thereof against a biomarker disclosed herein(e.g., CCL20, IL22, LCN2, FCP or CRP) is used as a “capture” antibody.The capture antibody is attached to a solid support, an antigen from asample or standard is allowed to bind to the capture antibody, and thena second antibody or antigen binding fragment thereof against the samebiomarker (e.g., CCL20, IL22, LCN2, FCP or CRP) comprising a detectablelabel, i.e., a “detection” antibody, is added. The detection antibodycan be detected either by an enzymatic reaction, an ECL reaction,radioactivity, or any other detection method known in the art.

In some aspects, the immunoassay comprises the following steps: First,the capture antibody or fragment thereof is allowed to bind to a solidsupport, e.g., a multi-well plate or other assay device known to thoseof ordinary skill in the art. The capture antibody is allowed to attachfor a period of time, e.g., overnight, and then unbound antibody isremoved. The plate can then be washed to remove any unbound captureantibody. The plate can then be treated with a blocking solution toallow non-specific protein to bind to any unbound regions of the solidsupport. Typical blocking solutions include an unrelated protein, e.g.,nonfat dry milk or serum albumin. The plate can then again be washed toremove any unbound blocking solution. Next, a sample suspected ofcontaining a biomarker disclosed herein (e.g., CCL20, IL22, LCN2, FCP orCRP) is added to the plate. Samples are typically serially diluted andplated in duplicate or triplicate. Controls, including standard amountsof biomarker (e.g., CCL20, IL22, LCN2, FCP or CRP) or a suitablefragment thereof and various negative controls are also included. Theantigen is allowed to bind to the capture antibody for a period of time,e.g., one hour at room temperature. Following incubation, the plate canthen be washed to remove any unbound antigen. Next, a detection antibodyis added. The detection antibody is typically an antibody thatspecifically binds to an epitope in the biomarker (e.g., CCL20, IL22,LCN2, FCP or CRP) than is different from the epitope to which thecapture antibody binds. The detection antibody can be labeled orunlabeled. Where the detection antibody is unlabeled, an addition stepof addition a labeled secondary antibody will be required, as is wellknown by those of ordinary skill in the art.

The detection antibody can be directly labeled with an enzyme, e.g.,horseradish peroxidase or alkaline phosphatase, or can be labeled with atag that will allow an enzyme to bind. For example the detectionantibody can be conjugated to biotin, and the enzyme attached in asubsequent step by allowing enzyme-conjugated streptavidin to bind tothe biotin tag. Alternatively, the detection antibody can be conjugatedto a chemiluminescent, fluorescent, or ECL tag. An example of the latteris a ruthenium chelate. Following incubation, the plate can then bewashed to remove any unbound detection antibody. Detection of thedetection antibody can be accomplished by methods that vary based on thetype of detection antibody that is used.

If the detection antibody is tagged with biotin, then enzyme-conjugatedstreptavidin is added, unbound streptavidin is washed away, and asubstrate is added which provides a colorimetric reaction that can beread, e.g., on a spectrophotometer. If the detection antibody isconjugated to a ruthenium chelate, the plate is subjected to electricalcurrent, and light emission is measured.

Immunoassays for detecting the biomarkers disclosed herein (e.g., CCL20,IL22, LCN2, FCP or CRP) can be either competitive or noncompetitive.Noncompetitive immunoassays are assays in which the amount of capturedanalyte is directly measured. In competitive assays, the amount ofanalyte in the sample is measured indirectly by measuring the amount ofan added (exogenous) labeled analyte displaced (or competed away) from acapture agent by the analyte present in the sample. In one competitiveassay, a known amount of, for example, labeled CCL20 is added to thesample, and the sample is then contacted with a capture agent. Theamount of labeled CCL20 bound to the antibody is inversely proportionalto the concentration of CCL20 present in the sample.

In some aspects, the method directly measures the levels of CCL20 (orany other biomarker disclosed herein) in a patient sample, whereabsolute levels are calculated by plotting the immunoassay results on astandard curve using, e.g., purified full length CCL20 or an CCL20fragment (or a full length biomarker disclosed herein or a fragmentthereof). The detected signal from the detection antibody can then bequantitated based on the various standards and controls included on theplate. By plotting the results on a standard curve, the absolute levelsof CCL20 (or any other biomarker disclosed herein) in the test samplescan be calculated, e.g., in pg or ng of biomarker per mL, or pg or ng ofbiomarker per mg of total protein.

Detection assays for the biomarkers disclosed herein (e.g., CCL20, IL22,LCN2, FCP or CRP) can be scored (as positive or negative or quantity ofanalyte) according to standard methods well known to those of skill inthe art. The particular method of scoring will depend on the assayformat and choice of label. For example, a Western Blot assay can bescored by visualizing the colored product produced by the enzymaticlabel. A clearly visible colored band or spot at the correct molecularweight is scored as a positive result, while the absence of a clearlyvisible spot or band is scored as a negative. The intensity of the bandor spot can provide a quantitative measure of analyte concentration.

In some aspects, the measured expression levels of the biomarkersdisclosed herein (e.g., CCL20, IL22, LCN2, FCP or CRP) represent anaverage expression level or a mean expression level based on more thanone measurement of the expression level. In some aspects, the measuredexpression level is an average or mean of several measurements ofexpression levels of the same sample. In some aspects, the measuredexpression level is an average or mean of several measurements ofexpression levels of different samples containing the same componentsobtained from the same subject. In some aspects, the measured expressionlevel is quantile normalized, as is done in RNA Seq techniques usingtechniques well known by those of ordinary skill in the art.

The term “level” as applied to a biomarker disclosed herein, e.g., as in“CCL20 level,” “IL22 level,” “LCN2 level,” “FCP level,” or “CRP level,”refers to a measurement that is made using any analytical method fordetecting presence or expression of the biomarker (protein expression orgene expression) in a biological sample and that indicates the presence,absence, absolute amount or concentration, relative amount orconcentration, titer, expression level, ratio of measured levels, or thelike, of, for, or corresponding to biomarker in the biological sample.

The exact nature of the “value” or “level” depends on the specificdesigns and components of the particular analytical method (e.g.,immunoassays, mass spectrometry methods, in vivo molecular imaging, geneexpression profiling, aptamer-based assays, etc.) employed to detect abiomarker disclosed herein (e.g., CCL20, IL22, LCN2, FCP or CRP). See,e.g., U.S. 2010/00221752.

As used herein with reference to the disclosed biomarkers (e.g., CCL20,IL22, LCN2, FCP or CRP), the terms “elevated level,” “increased level,”“higher level,” or “high level” refer to a level in a biological sample(e.g., blood serum) that is higher than the expression level or range ofthe biomarker measured in a control sample (“normal level”), or aspecified threshold disclosed herein. These thresholds include, e.g.,about 22.6 pg/mL for CCL20 serum protein as measured using a CCL20immunoassay, including the CCL20 immunoassay described in Example 3;about 15.6 pg/mL for IL22 serum protein as measured using an IL22immunoassay, including the IL22 immunoassay described in Example 4;about 215 ng/mL for LCN2 serum protein as measured using an LCN2immunoassay, including the LCN2 immunoassay described in Example 4.

As used herein with reference to the biomarkers disclosed herein (e.g.,CCL20, IL22, LCN2, FCP or CRP), the terms “lowered level,” “reducedlevel,” “decreased level,” or “low level” refer to a level in abiological sample (e.g., blood serum) that is lower than the expressionlevel or range of the biomarker measured in a control sample (“normallevel”), or a specified threshold disclosed herein. These thresholdsinclude, e.g., about 22.6 pg/mL for CCL20 serum protein as measuredusing a CCL20 immunoassay, including the CCL20 immunoassay described inExample 3; about 15.6 pg/mL for IL22 serum protein as measured using anIL22 immunoassay, including the IL22 immunoassay described in Example 4;about 215 ng/mL for LCN2 serum protein as measured using an LCN2immunoassay, including the LCN2 immunoassay described in Example 4.

The normal level or range for a biomarker disclosed herein (e.g., CCL20,IL22, LCN2, FCP and/or CRP) can be defined in accordance with standardpractice. Thus, the level measured in a particular biological sample canbe compared with level or range of levels determined in similar normalsamples. In this context, a normal sample or a control sample would be,for example, a sample obtained from an individual with no detectablesymptoms of an IL23-mediated disease or disorder. Thus, the level of,e.g., CCL20, is said to be a low level, lowered level, reduced level,decreased level, or grammatical variants thereof when the level of CCL20is present in the test sample at a lower level or range than in a normalsample, control sample, or a specific threshold level disclosed herein.These thresholds include, e.g., about 22.6 pg/mL for CCL20 serum proteinas measured using a CCL20 immunoassay, including the CCL20 immunoassaydescribed in Example 3; about 15.6 pg/mL for IL22 serum protein asmeasured using an IL22 immunoassay, including the IL22 immunoassaydescribed in Example 4; about 215 ng/mL for LCN2 serum protein asmeasured using an LCN2 immunoassay, including the LCN2 immunoassaydescribed in Example 4.

In some aspects, the level of a biomarker disclosed herein (e.g., CCL20,IL22, LCN2, FCP and/or CRP) is considered to be elevated or high if itis at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%,125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%,185%, 190%, 195%, 200%, 205%, 210%, 215%, 220%, 225%, 230%, 235%, 240%,245%, 250%, 255%, 260%, 265%, 270%, 275%, 280%, 285%, 290%, 295%, 300%,305%, 310%, 315%, 320%, 325%, 330%, 335%, 340%, 345%, 350%, 355%,360{circumflex over ( )}, 365%, 370%, 375%, 380%, 385%, 390%, 395%,400%, 405%, 410%, 415%, 420%, 425%, 420%, 435%, 440%, 445%, 450%, 455%,460%, 465%, 470%, 475%, 480%, 485%, 490%, 495%, or 500% higher than anormal sample or control sample, or a specific threshold level disclosedherein.

In some aspects, the level of a biomarker disclosed herein (e.g., CCL20,IL22, LCN2, FCP and/or CRP) is considered to be reduced or low if it isat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%,125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%,185%, 190%, 195%, 200%, 205%, 210%, 215%, 220%, 225%, 230%, 235%, 240%,245%, 250%, 255%, 260%, 265%, 270%, 275%, 280%, 285%, 290%, 295%, 300%,305%, 310%, 315%, 320%, 325%, 330%, 335%, 340%, 345%, 350%, 355%,360{circumflex over ( )}, 365%, 370%, 375%, 380%, 385%, 390%, 395%,400%, 405%, 410%, 415%, 420%, 425%, 420%, 435%, 440%, 445%, 450%, 455%,460%, 465%, 470%, 475%, 480%, 485%, 490%, 495%, or 500% lower than anormal sample or control sample, or a specific threshold level disclosedherein.

As used herein, the term “threshold level” (or alternatively herein“threshold value” or “predetermined threshold level”) refers to a levelof a biomarker disclosed herein (e.g., CCL20, IL22, LCN2, FCP and/orCRP) which may be of interest for comparative purposes. In some aspects,a threshold level may be the expression level of a protein or nucleicacid expressed as an average of the level of the expression level of aprotein or nucleic acid from samples taken from a control population ofhealthy (disease-free) subjects. In some aspects, the threshold levelmay be the level in the same subject at a different time, e.g., beforethe present assay, such as the level determined prior to the subjectdeveloping the disease or prior to initiating therapy. In general,samples are normalized by a common factor. For example, body fluidsamples are normalized by volume body fluid and cell-containing samplesare normalized by protein content or cell count. In another aspect, thethreshold level may also refer to the level of expression of the samebiomarker in a corresponding control sample or control group of subjectswhich do not respond to treatment, e.g., with an IL23 antagonist (e.g.,an anti-IL23 antibody targeting, e.g., the p19 subunit of IL23, or anantigen-binding fragment thereof).

In some aspects, the expression level of a biomarker disclosed herein(e.g., CCL20, IL22, LCN2, FCP or CRP) is compared to a threshold level(or alternatively herein a “predetermined threshold level”). Thus, asused herein, the term “threshold level” or “predetermined thresholdlevel” is a cutoff or threshold against which the measured expressionlevel of a protein or nucleic acid is compared.

Based on comparison to known control samples, a “threshold level” for abiomarker disclosed herein (e.g., CCL20, IL22, LCN2, FCP or CRP) can bedetermined, and test samples that fall above or below the biomarker'sthreshold levels indicate that the patient from whom the sample wasobtained may or may not benefit from treatment with an IL23 antagonist(e.g., an anti-IL23 antibody targeting, e.g., the p19 subunit of IL23,or an antigen-binding fragment thereof). For example, for CCL20, samplesfalling below the threshold level for CCL20 would indicate that thepatient may benefit from treatment with an IL23 antagonist. Conversely,samples falling above the threshold level for CCL20 would indicate thatthe patient may not benefit from treatment with an IL23 antagonist. Forexample, for IL22 or LCN2, samples falling above the threshold level forIL22 or LNC2 would indicate that the patient may benefit from treatmentwith an IL23 antagonist. Conversely, samples falling below the thresholdlevel for IL22 or LCN2 would indicate that the patient may not benefitfrom treatment with an IL23 antagonist.

In some aspects, threshold levels (e.g., protein expression levels orgene expression levels) for a biomarker disclosed herein (e.g., CCL20,IL22, LCN2, FCP or CRP) can be predetermined and matched as to the typeof sample (e.g., serum, lung tissue, skin), the type of disease (e.g.,asthma, IPF, COPD, Crohn's Disease, UC, or atopic dermatitis), and insome instances, the assay used.

As described in the Examples, CCL20 protein levels quantified using theCCL20 immunoassay described in Example 3 from serum samples from apopulation of moderate to severe Crohn's disease patients indicated thatpatients having CCL20 levels lower than or equal to 22.6 pg/mL (themedian CCL20 level for the population of patients in the study) hadincreased clinical responses to an anti-IL23 antibody, while patientswith CCL20 levels higher than 22.6 pg/mL had reduced clinical responsessimilar to those in the placebo group. Accordingly, in some aspects ofthe methods disclosed herein, the predetermined CCL20 threshold level isabout 22.6 pg/mL CCL20 protein expression in serum as measured using theCCL20 immunoassay described in Example 3. In some other aspects, thepredetermined CCL20 threshold level is about the median CCL20 value inserum measured from a plurality of patients having an IL23-mediateddisease as measured using the CCL20 immunoassay described in Example 3.In some aspects, a “low level of CCL20” (CCL20 LO) is defined as a valuebelow the median value of about 22.6 pg/mL CCL20 protein expression inserum as measured using the CCL20 immunoassay described in Example 3. Insome aspects, a “high level of CCL20 (CCL20 HI) is defined as a valueequal to or above the median value of about 22.6 pg/mL CCL20 proteinexpression in serum as measured using the CCL20 immunoassay described inExample 3.

In some aspects, the predetermined CCL20 threshold level is about 22.6pg/mL+/−10 pg/mL CCL20 protein expression in serum as measured using theCCL20 immunoassay described in Example 3. Accordingly, the CCL20threshold level can be about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 pg/mL. In some aspects a“low level of CCL20” (CCL20 LO) is defined as a value below one of thesethreshold levels, whereas a “high level of CCL20” (CCL20 HI) is definedas a value equal to or above the same threshold level (i.e., if thethreshold level was 20 pg/mL, a low level of CCL20 would be below 20pg/mL, and a high level of CCL20 would be 20 pg/mL or above).

In some aspects, the predetermined CCL20 threshold corresponds to the1^(st), 2^(nd), 3^(rd), 4^(th), 5^(th), or 6^(th), decile CCL20 baselinelevel as reported in TABLE 6. In some aspects, the predetermined CCL20threshold level is about 11.8 pg/mL, 14.5 pg/mL, about 16.3 pg/mL, about20.8 pg/mL, about 22.6 pg/mL, about 26.5 pg/mL, or about 32.1 pg/mLCCL20 protein expression in serum as measured using the CCL20immunoassay described in Example 3. In some aspects a “low level ofCCL20” (CCL20 LO) is defined as a value below one of these thresholdlevels, whereas a “high level of CCL20” (CCL20 HI) is defined as a valueequal to or above the same threshold level.

In some aspects, the predetermined CCL20 threshold level as measuredusing the CCL20 immunoassay described in Example 3 corresponds to aconcentration of CCL20 in serum between 5 pg/mL and 55 pg/mL, e.g.,about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 pg/mL.

As described in the Examples, IL22 protein levels quantified using theIL22 immunoassay described in Example 5 from serum samples from apopulation of moderate to severe Crohn's disease patients indicated thatpatients having higher or elevated IL22 levels greater than or equal to15.6 pg/mL (the median IL22 level for the population of patients in thestudy) had increased clinical responses to an anti-IL23 antibody, whilepatients with lower or reduced IL22 levels less than 15.6 pg/mL hadreduced clinical responses similar to those in the placebo group.Accordingly, in some aspects of the methods disclosed herein, thepredetermined IL22 threshold level is about 15.6 pg/mL IL22 proteinexpression in serum as measured using the IL22 immunoassay described inExample 4. In some other aspects, the predetermined IL22 threshold levelis about the median IL22 value in serum measured from a plurality ofpatients having an IL23-mediated disease as measured using the IL22immunoassay described in Example 4. In some aspects, a “low level ofIL22” (IL22 LO) is defined as a value below the median value of about15.6 pg/mL IL22 protein expression in serum as measured using the IL22immunoassay described in Example 4. In some aspects, a “high level ofIL22” (IL22 HI) is defined as a value equal to or above the median valueof about 15.6 pg/mL IL22 protein expression in serum as measured usingthe IL22 immunoassay described in Example 4.

In some aspects, the predetermined IL22 threshold level is about 15.6pg/mL+/−10 pg/mL IL22 protein expression in serum as measured using theIL22 immunoassay described in Example 4. Accordingly, the IL22 thresholdlevel can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25 or 26 pg/mL. In some aspects a “low level ofIL22” (IL22 LO) is defined as a value below one of these thresholdlevels, whereas a “high level of IL22” (IL22 HI) is defined as a valueequal to or above the same threshold level (i.e., if the threshold levelwas 20 pg/mL, a low level of IL22 would be below 20 pg/mL, and a highlevel of IL22 would be 20 pg/mL or above).

In some aspects, the predetermined IL22 threshold corresponds to the2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th) or 8^(th) decile IL22baseline level as reported in TABLE 4. In some aspects, thepredetermined IL22 threshold level is about 7.9 pg/mL, about 11.3 pg/mL,about 12.7 pg/mL, about 15.6 pg/mL, about 19.6 pg/mL, about 23.1 pg/mL,about 31.4 pg/mL or about 46.8 pg/mL IL22 protein expression in serum asmeasured using the IL22 immunoassay described in Example 4. In someaspects a “low level of IL22” (IL22 LO) is defined as a value below oneof these threshold levels, whereas a “high level of IL22” (IL22 HI) isdefined as a value equal to or above the same threshold level.

In some aspects, the predetermined IL22 threshold level as measuredusing the IL22 immunoassay described in Example 4 corresponds to aconcentration of IL22 in serum between 7.9 pg/mL and 31.4 pg/mL, e.g.,about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, or 31 pg/mL.

As described in the Examples, LCN2 protein levels quantified using theLCN2 immunoassay described in Example 4 from serum samples from apopulation of moderate to severe Crohn's disease patients indicated thatpatients having higher or elevated LCN2 levels greater than or equal to215 ng/mL (the median LCN2 level for the population of patients in thestudy) had increased clinical responses to an anti-IL23 antibody, whilepatients with lower or reduced LCN2 levels less than 215 ng/mL hadreduced clinical responses. Accordingly, in some aspects of the methodsdisclosed herein, the predetermined LCN2 threshold level is about 215ng/mL LCN2 protein expression in serum as measured using the LCN2immunoassay described in Example 4. In some other aspects, thepredetermined LCN2 threshold level is about the median LCN2 value inserum measured from a plurality of patients having an IL23-mediateddisease as measured using the LCN2 immunoassay described in Example 4.In some aspects, a “low level of LCN2” (LCN2 LO) is defined as a valuebelow the median value of about 215 ng/mL LCN2 protein expression inserum as measured using the LCN2 immunoassay described in Example 4. Insome aspects, a “high level of LCN2” (LCN2 HI) is defined as a valueequal to or above the median value of about 215 ng LCN2/mL LCN2 proteinexpression in serum as measured using the LCN2 immunoassay described inExample 4.

In some aspects, the predetermined LCN2 threshold level is about 215ng/mL+/−70 ng/mL LCN2 protein expression in serum as measured using theLCN2 immunoassay described in Example 4. Accordingly, the LCN2 thresholdlevel can be about 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260,265, 270, 275, 280 or 285 ng/mL. In some aspects a “low level of LCN2”(LCN2 LO) is defined as a value below one of these threshold levels,whereas a “high level of LCN2” (LCN2 HI) is defined as a value equal toor above the same threshold level (i.e., if the threshold level was 250ng/mL, a low level of LCN2 would be below 250 ng/mL, and a high level ofLCN2 would be 250 ng/mL or above).

In some aspects, the predetermined LCN2 threshold corresponds to the1^(st), 2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th) or 7^(th) decile LCN2baseline level as reported in TABLE 5. In some aspects, thepredetermined LCN2 threshold level is about 142.8 ng/mL, about 163.6ng/mL, about 184.3 ng/mL, about 201.3 ng/mL, about 214.6 ng/mL, about233.4 ng/mL, about 261.1 ng/mL, about 294.8 ng/mL, or about 326.6 ng/mLLCN2 protein expression in serum as measured using the immunoassaydescribed in Example 4. In some aspects a “low level of LCN2” (LCN2 LO)is defined as a value below one of these threshold levels, whereas a“high level of LCN2” (LCN2 HI) is defined as a value equal to or abovethe same threshold level.

In some aspects, the predetermined LCN2 threshold level as measuredusing the LCN2 immunoassay described in Example 4 corresponds to aconcentration of LCN2 in serum between 143 ng/mL and 261 ng/mL, e.g.,about 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205,210, 215, 220, 225, 230, 235, 240, 245, 250, 255 or 260 ng/mL.

In some aspects, the threshold level is the average or mean expressionlevel measured from samples obtained from healthy volunteers as reportedin the manufacturer's manual of a commercial immunoassay used to detectthe presence or absence of biomarkers disclosed herein (e.g., CCL20,IL22, LCN2, FCP or CRP) in a sample.

A solid phase sandwich ELISA kit to quantify CCL20 is available from R&DSystems (Human CCL20/MIP-3 alpha Quantikine® ELISA Kit, Cat. # DM3A00).Other biomarkers disclosed herein can also be quantified using other R&DSystem's Quantikine® ELISA Kits namely IL22 (Human IL22 Quantikine®ELISA Kit, Cat. # D2200), LCN2 (Human Lipocalin-2/NGAL Quantikine® ELISAKit, Cat. # DLCN20) and CRP (Human C-Reactive Protein/CRP QuantikineELISA Kit, Cat. # DCRP00). The R&D Systems manufacturer's manuals forthe Human CCL20/MIP-3 alpha Quantikine ELISA Kit, Human IL22 Quantikine®ELISA Kit, the Human Lipocalin-2/NGAL Quantikine® ELISA Kit, and HumanC-Reactive Protein/CRP Quantikine ELISA Kit are herein incorporated byreference in their entireties. There are numerous tests available toquantify fecal calprotectin. See Labaere et al. United EuropeanGastroenterology Journal 2:30-37 (2014) for a review and comparison ofsix commercial fecal calprotectin tests: three rapidimmunochromatographic assays (Buhlmann Quantum Blue, Eurospital Calfast,and Biotec Certest), two ELISAs (Eurospital and Calprolab), and oneautomated immunoassay (Phadia EliA).

As indicated in the Human CCL20 QUANTIKINE® Assay manufacturer's manual,detectable CCL20 levels in serum (n=74), EDTA plasma (n=34), and heparinplasma (n=34) from apparently healthy volunteers were evaluated usingthe CCL20 QUANTIKINE® immunoassay and CCL20 mean values were 26.3 pg/mLfor serum, 14.3 pg/mL for EDTA plasma, and 14.2 pg/mL for heparinplasma, respectively. Accordingly, in some aspects of the methodsdisclosed herein, the predetermined CCL20 threshold level is about 26.3pg/mL for serum, about 14.3 pg/mL for EDTA plasma, and about 14.2 pg/mLfor heparin plasma as measured by the Human CCL20/MIP-3 alphaQUANTIKINE® ELISA Kit (Catalog Number DM3A00); R&D Systems) followingthe manufacturer's instructions, for their respective samples.

Similarly, as indicated in the Human IL22 QUANTIKINE® Assaymanufacturer's manual, IL22 levels in serum (n=53), EDTA plasma (n=51),heparin plasma (n=53) and urine (n=42) from apparently healthyvolunteers were evaluated using the IL22 QUANTIKINE® immunoassay and theIL22 mean values were 35.7 pg/mL for serum, 29.3 pg/mL for EDTA plasma,31.7 pg/mL for heparin plasma, and 35.2 pg/mL for urine, respectively.Accordingly, in some aspects of the methods disclosed herein, thepredetermined IL22 threshold level is about 35.7 pg/mL for serum, about29.3 pg/mL for EDTA plasma, about 31.7 pg/mL for heparin plasma, and/orabout 35.2 pg/mL for urine as measured by the Human IL22 QUANTIKINE®ELISA Kit (Catalog Number D2200; R&D Systems) following themanufacturer's instructions, for their respective samples.

As indicated in the Human LCN2 QUANTIKINE® Assay manufacturer's manual,detectable LCN2 levels in serum (n=35), heparin plasma (n=35), saliva(n=9) and urine (n=19) from apparently healthy volunteers were evaluatedusing the LCN2 QUANTIKINE® immunoassay and the LCN2 mean values were 119ng/mL for serum, 94 ng/mL for heparin plasma, 320 ng/mL for saliva, and9.94 ng/mL for urine, respectively. Accordingly, in some aspects of themethods disclosed herein, the predetermined LCN2 threshold level isabout 119 ng/mL for serum, about 94 ng/mL for heparin plasma, about 320ng/mL for saliva, and about 9.94 ng/mL for urine as measured by theHuman Lipocalin-2/NGAL QUANTIKINE® ELISA Kit (Catalog NumberDLCN20/SLCN20/PDLCN20; R&D Systems) following the manufacturer'sinstructions, for their respective samples.

In some aspects, the expression level of a biomarker disclosed herein(e.g., CCL20, IL22, LCN2, CRP, and/or FCP) measured in the sample isabove or below the threshold level or threshold value. In these aspectswhere the expression level of a biomarker disclosed herein (e.g., CCL20,IL22, LCN2, CRP, and/or FCP) measured in the sample is above or belowthe threshold level or threshold, the expression level can indicate thatthe patient from whom the sample was taken may benefit or not fromtreatment with an IL23 antagonist (e.g., an anti-IL23 antibodytargeting, e.g., the p19 subunit of IL23, or an antigen-binding fragmentthereof). The extent to which the measured expression level is above orbelow the threshold level or threshold value may be to any extent.

In exemplary aspects, the measured expression level of a biomarkerdisclosed herein (e.g., CCL20, IL22, LCN2, CRP, and/or FCP) is at leastor about 10% greater or lower than the threshold level, e.g., at leastor about 15% greater or lower than the threshold level, at least orabout 20% greater or lower than the threshold level, at least or about25% greater or lower than the threshold level, at least or about 30%greater or lower than the threshold level, at least or about 35% greateror lower than the threshold level, at least or about 40% greater orlower than the threshold level, at least or about 45% greater or lowerthan the threshold level, at least or about 50% greater or lower thanthe threshold level, at least or about 55% greater or lower than thethreshold level, at least or about 60% greater or lower than thethreshold level, at least or about 65% greater or lower than thethreshold level, at least or about 70% greater or lower than thethreshold level, at least or about 75% greater or lower than thethreshold level, at least or about 80% greater or lower than thethreshold level, at least or about 85% greater or lower than thethreshold level, at least or about 90% greater or lower than thethreshold level, at least or about 95% greater or lower than thethreshold level.

In exemplary aspects, the measured expression level of a biomarkerdisclosed herein (e.g., CCL20, IL22, LCN2, CRP, and/or FCP) is at least2-fold greater or lower than the threshold level, at least 3-foldgreater or lower than the threshold level, at least 4-fold greater orlower than the threshold level, at least 5-fold greater or lower thanthe threshold level, at least 6-fold greater or lower than the thresholdlevel, at least 7-fold greater or lower than the threshold level, atleast 8-fold greater or lower than the threshold level, at least 9-foldgreater or lower than the threshold level, or at least 10-fold greateror lower than the threshold level.

As discussed above, the level of a biomarker disclosed herein (e.g.,CCL20, IL22, LCN2, CRP, and/or FCP) can be determined using methodsknown in the art. A person skilled in the art would appreciate that inaddition to the QUANTIKINE® assays disclosed above, there are numerousmethods available in the art that would allow the skilled artisan todetermine threshold levels as described throughout this section,including the methods and immunoassays described in the Examples.

In some aspects, the predetermined threshold level of a biomarkerdisclosed herein (e.g., CCL20, IL22, LCN2, CRP, and/or FCP) is definedwith respect to a certain percentile value in a population of subjects(e.g., a plurality of normal healthy patients, patients with anon-IL23-mediated disease and/or patients with an IL23-mediateddisease).

In some aspects, the predetermined threshold level for CCL20 correspondsto the 10^(th), 15^(th), 20^(th), 25^(th), 30^(th), 35^(th), 40^(th),45^(th), 50^(th), 50^(th), 55^(th), 60^(th), 65^(th), 70^(th), 75^(th),80^(th), 85^(th), or 90^(th) percentile.

Similarly, in some aspects, the predetermined threshold level for IL22corresponds to the 10^(th), 15^(th), 20^(th), 25^(th), 30^(th), 35^(th),40^(th), 45^(th), 50^(th), 50^(th), 55^(th), 60^(th), 65^(th), 70^(th),75^(th), 80^(th), 85^(th), or 90^(th) percentile; the predeterminedthreshold level for LCN2 corresponds to the 10^(th), 15^(th), 20^(th),25^(th), 30^(th), 35^(th), 40^(th), 45^(th), 50^(th), 50^(th), 55^(th),60^(th), 65^(th), 70^(th), 75^(th), 80^(th), 85^(th), or 90^(th)percentile; the predetermined threshold level for CRP corresponds to the10^(th), 15^(th), 20^(th), 25^(th), 30^(th), 35^(th), 40^(th), 45^(th),50^(th), 50^(th), 55^(th), 60^(th), 65^(th), 70^(th), 75^(th), 80^(th),85^(th), or 90^(th) percentile; and/or the predetermined threshold levelfor FCP corresponds to the 10^(th), 15^(th), 20^(th), 25^(th), 30^(th),35^(th), 40^(th), 45^(th), 50^(th), 50^(th), 55^(th), 60^(th), 65^(th),70^(th), 75^(th), 80^(th), 85^(th), or 90^(th) percentile.

The threshold level (e.g., a protein expression level or a geneexpression level) of a biomarker disclosed herein (e.g., CCL20, IL22,LCN2, CRP, and/or FCP) can vary based on the nature of the assay, e.g.,the capture and detection antibodies used, the source, purity, andcomposition of the standard, and the like.

In one aspect, instead of using an arbitrary threshold level todetermine whether a patient can benefit from treatment with an IL23antagonist (e.g., an anti-IL23 antibody targeting, e.g., the p19 subunitof IL23, such as MEDI2070, or an antigen-binding fragment thereof), thepatient's level of a biomarker disclosed herein (e.g., CCL20, IL22,LCN2, CRP, and/or FCP) can be compared to one or more control levels.According to this aspect, the test sample (e.g., a sample from a patientsuffering from an IL23-mediated disease or disorder) is compared to oneor more control samples (e.g., samples taken from normal healthyindividuals, earlier samples taken from the same patient, samples takenfrom patients with a non-IL23-mediated subset of the patient's disease,e.g., asthma, COPD, IPF, Crohn's Disease, UC, or atopic dermatitis, apre-determined standard amount of isolated biomarker protein or gene ora fragment thereof, or a combination thereof).

The results can be expressed as a ratio with the control samples todetermine a percent increase or a percent decrease in the patient'sbiomarker levels (e.g., a protein expression level or a gene expressionlevel) compared to the control biomarker levels. The control sample canbe a matched pair with the patient sample, e.g., one or more of wholeblood if the patient sample is whole blood, serum if the patient sampleis serum, plasma if the patient sample is plasma, saliva if the patientsample is saliva, urine if the patient sample is urine, sputum if thepatient sample is sputum, bronchoalveolar lavage fluid if the patientsample is bronchoalveolar lavage fluid, lung tissue if the patientsample is lung tissue, or skin if the patient sample is skin. Oncedetermined, a biomarker expression level can be recorded in a patient'smedical record.

In a particular aspect, a low level of CCL20 (lower that about 22.6pg/mL in serum as measured using the CCL20 immunoassay disclosed inExample 3) is predictive of positive clinical response to an IL23antagonist (e.g., an anti-IL23 antibody targeting, e.g., the p19 subunitof IL23, such as MEDI2070, or an antigen-binding fragment thereof) in apatient having an IL23-mediated disease (including, e.g., CD). In oneaspect, the high level of CCL20 is a value between about 12 pg/mL andabout 55 pg/mL as measured using the CCL20 immunoassay disclosed inExample 3.

In some aspects, as disclosed above, CCL20 can be combined with otherbiomarkers such as IL22, LCN2, FCP and/or CRP. In a particular aspect,CCL20 can be combined with IL22 and/or LCN2. Accordingly, in someaspects, a high level of IL22 (at least about 15.6 pg/mL in serum asmeasured using the IL22 immunoassay disclosed in Example 4), and/or ahigh level of LCN2 (at least about 215 ng/mL as measured using the LCN2immunoassay disclosed in Example 4), combined with CCL20 values belowthe threshold levels disclosed above, are predictive of positiveclinical response to an IL23 antagonist (e.g., an anti-IL23 antibodytargeting, e.g., the p19 subunit of IL23, such as MEDI2070, or anantigen-binding fragment thereof) in a patient having an IL23-mediateddisease (including, e.g., CD).

In one aspect, the high level of IL22 is a value between about 7.9 pg/mLand about 31.4 pg/mL as measured using the IL22 immunoassay disclosed inExample 4. In one aspect, the high level of LCN2 is a value betweenabout 143 pg/mL and about 261 pg/mL as measured using the LCN2immunoassay disclosed in Example 4.

In a particular aspect, a level of CCL20 below about 11.8 pg/mL, about14.5 pg/mL, about 16.3 pg/mL, about 20.8 pg/mL, about 22.6 pg/mL, about26.5 pg/mL, or about 32.1 pg/mL CCL20 protein expression in serum asmeasured using the CCL20 immunoassay described in Example 3 ispredictive of positive clinical response to an IL23 antagonist (e.g., ananti-IL23 antibody targeting, e.g., the p19 subunit of IL23, such asMEDI2070, or an antigen-binding fragment thereof) in a patient having anIL23-mediated disease (including, e.g., CD).

In a particular aspect, the CCL20 disclosed above combined with a levelof IL22 above: about 7.9 pg/mL, about 11.3 pg/mL, about 12.7 pg/mL,about 15.6 pg/mL, about 19.6 pg/mL, about 23.1 pg/mL, about 31.4 pg/mLor about 49.8 pg/mL IL22 protein expression in serum as measured usingthe IL22 immunoassay described in Example 4; and/or a level of LCN2above: about 142.8 ng/mL, about 163.6 ng/mL, about 184.3 ng/mL, about201.3 ng/mL, about 214.6 ng/mL, about 233.4 ng/mL, about 261.1 ng/mL,about 294.8 ng/mL, or about 326.6 ng/mL LCN2 expression as measuredaccording to the LCN2 immunoassay disclosed in Example 4 are predictiveof positive clinical response to an IL23 antagonist (e.g., an anti-IL23antibody targeting, e.g., the p19 subunit of IL23, such as MEDI2070, oran antigen-binding fragment thereof) in a patient having anIL23-mediated disease (including, e.g., CD).

In one aspect, administration of the IL23 antagonist (e.g., an anti-IL23antibody targeting, e.g., the p19 subunit of IL23, such as MEDI2070, oran antigen-binding fragment thereof) results in a Crohn's DiseaseActivity Index (CDAI) response score reduction of at least 100 points,or reduction of the total CDAI score to below 150 points after firstadministering the IL23 antagonist (e.g., an anti-IL23 antibodytargeting, e.g., the p19 subunit of IL23, such as MEDI2070, or anantigen-binding fragment thereof).

In other aspects, the administration of the IL23 antagonist (e.g., ananti-IL23 antibody targeting, e.g., the p19 subunit of IL23, such asMEDI2070, or an antigen-binding fragment thereof) results in a Crohn'sDisease Activity Index (CDAI) response score reduction of at least 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 points afteradministering of one or more doses the anti-IL23 antibody orantigen-binding fragment thereof.

IV. IL23 Antagonists

CCL20, alone or combined with other IL23 pathway biomarkers disclosedherein (e.g., IL22 and/or LCN2), and/or in combination with otherbiomarkers indicative of inflammation (e.g., FCP and/or CRP) can beused, for example, to determine whether to treat, select for treatment,monitor the treatment, or a begin, modify, or cease the treatment of apatient suffering from an IL23-mediated disease or disorder with an IL23antagonist (e.g., an anti-IL23 antibody targeting, e.g., the p19 subunitof IL23, such as MEDI2070, or an antigen-binding fragment thereof).

As used herein, the term “IL23 antagonist” refers to any agent that canaffect the expression, activity, or half-life of IL23 either in vitro orin vivo, or symptoms, pathology, or sequelae caused by or exacerbated byIL23 in a subject with an IL23-mediated disease or disorder, e.g., CD.An IL23 antagonist can be any therapeutic agent as defined herein, whicheither directly or indirectly can inhibit, lessen, or neutralize IL23activity, inhibit or reduce IL23 expression, reduce IL23 half-life, orcan prevent exacerbation of symptoms due to IL23. In certain aspects, anIL23 antagonist is an anti-IL23 monoclonal antibody.

Specific IL23 antagonists contemplated herein specifically bind andinhibit IL23, but do not inhibit IL12. An IL23 antagonist or bindingagent of the present disclosure (e.g., and antibody such as MEDI2070)competitively inhibits binding of a reference molecule (e.g., adifferent IL23 antagonist or binding agent) to a given target site if itpreferentially binds to that target site to the extent that it blocks,to some degree, binding of the reference molecule to the target site.Competitive inhibition can be determined by any method known in the art,for example, competition ELISA assays. An IL23 antagonist or bindingagent of the present disclosure (e.g., an antibody such as MEDI2070) canbe said to competitively inhibit binding of the reference molecule to agiven epitope by at least about 90%, at least about 85%, at least about80%, at least about 75%, at least about 70%, at least about 65%, atleast about 60%, at least about 55%, or at least 50%.

In some aspects, the IL23 antagonist is an anti-IL23 antibody targeting,e.g., the p19 subunit of IL23, the p40 subunit of IL23, or both. Anexemplary IL23 antagonist contemplated is the anti-IL23 antibodyMEDI2070, which targets the p19 subunit of IL23. MEDI2070 is a fullyhuman, Chinese Hamster Ovary (CHO) cell-derived, immunoglobulin G2(IgG2) monoclonal antibody (MAb) that specifically binds to human IL23with high affinity and prevents IL23 from interacting with the IL23receptor. The molecule is a heterotetramer, consisting of 2 heavy chainsof the IgG2 subclass and 2 light chains of the lambda subclass, whichare covalently linked through disulfide bonds. See International Publ.WO 2011/056600, herein incorporated by reference.

MEDI2070 comprises a Heavy Chain of SEQ ID NO: 17 and a Light Chain ofSEQ ID NO: 18. MEDI2070 comprises a heavy chain variable region (SEQ IDNO: 7) comprising VH-CDR1, VH-CDR2 and VH-CDR3 with sequencescorresponding to SEQ ID NOs: 33, 34 and 35, respectively. MEDI2070comprises a light chain variable region (SEQ ID NO: 8) comprisingVL-CDR1, VL-CDR2 and VL-CDR3 with sequences corresponding to SEQ ID NOS:36, 37, and 38, respectively.

As used herein, the term “MEDI2070” refers not only to an intactMEDI2070 immunoglobulin, but also to MEDI2070 antigen-binding fragments,variant, or derivatives thereof, antibodies or fragments thereof thatbind to the same IL23 epitope as MEDI2070, or an antibodies or fragmentsthereof that competitively inhibit binding of MEDI2070 to IL23.Antibodies (or fragments thereof) that are identical or similar toMEDI2070 in amino acid sequence, particularly in the variable regions,or in the CDRs thereof (however, variations in the constant regions arealso contemplated) are contemplated. For example, in one aspect,MEDI2070 refers to a polypeptide having an amino acid sequence that isabout 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about95%, about 98%, about 99% or 100% identical to that of an MEDI2070polypeptide disclosed herein (the heavy chain of MEDI2070, the light ofMEDI2070). In some aspects, MEDI2070 is an isolated IL23 specificantigen binding protein comprising at least one, two, three, four, orthe six complementarity determining regions of MEDI2070.

Another exemplary IL23 antagonist contemplated is an anti-IL23 antibodywhich targets the p19 subunit of IL23 comprising a VH of SEQ ID NO: 45and/or a VL of SEQ ID NO: 46. In some aspects, the IL23 antagonistcomprises a heavy chain variable region comprising VH-CDR1, VH-CDR2 andVH-CDR3 from SEQ ID NO: 45 (i.e., SEQ ID NOs: 47-49), and/or a lightchain variable region comprising VL-CDR1, VL-CDR2 and VL-CDR3 from SEQID NO: 46 (i.e., SEQ ID NOs: 50-52). In some aspects, the IL23antagonist binds to the same IL23 epitope as an antibody comprising a VHof SEQ ID NO: 45 and a VL of SEQ ID NO: 46. In some aspects, the IL23antagonist competitively inhibits binding of an antibody comprising a VHof SEQ ID NO: 45 and a VL of SEQ ID NO: 46 to IL23. Antibodies (orfragments thereof) that comprise a VH and/or a VL domain identical orsimilar in amino acid sequence to a VH of SEQ ID NO: 45 and/or a VL ofSEQ ID NO: 46, particularly in the variable regions, or in the CDRsthereof (however, variations in the constant regions are alsocontemplated) are contemplated. For example, in one aspect, the IL23antagonist is a polypeptide having an amino acid sequence that is about70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%,about 98%, about 99% or 100% identical to a VH of SEQ ID NO:45 and/or aVL of SEQ ID NO:46. In some aspects, the IL23 antagonist an isolatedIL23 specific antigen binding protein comprising at least one, two,three, four, or the six complementarity determining regions of a VH ofSEQ ID NO:45 and/or a VL of SEQ ID NO: 46.

The term “IL23 antagonist” encompasses also the antibodies targetingIL23 described for example in U.S. Pat. Nos. 7,491,391, 7,807,414,7,872,102, 7,807,160, U.S. Pat. Nos. 8,362,212, 7,935,344; or U.S. Pat.No. 7,790,862; U.S. Application. Publ. Nos. US2012282269, US20090123479,US20120128689, or US2012264917: or International Publ. Nos. WO199905280,WO20070244846, WO2007027714, WO2007076524, WO2007147019, WO2008103473,WO2008103432, WO2009043933, WO2009082624, or WO 12009760, all of whichare herein incorporated by reference in their entireties. In someaspects, “IL23 antagonist” encompasses antibodies and antigen fragmentsthereof (including, e.g., constructs such as scFvs) comprising one, two,three, four, five or six of the CDR sequences of the antibodiesdisclosed in the cited references. In some aspects, “IL23 antagonist”encompasses antibodies and antigen fragments thereof (including, e.g.,constructs such as scFvs) competing for binding to IL23 with theantibodies disclosed in the cited references.

In some specific aspects, the term “IL23 antagonist” refers toustekinumab (CNTO-1275, STELARA®) (SEQ ID NOS: 19, 20), briakinumab(ABT-874) (SEQ ID NOS: 27, 28), Guselkumab (CNTO-1959), tildrakinumab(MK-3222; SCH-900222) (SEQ ID NOS: 29, 30), BI-655066 (see Krueger etal. J. Allergy Clin Immunol. 136:116-124 (2015)), LY-3074828 (see Gaffenet al. Nature Reviews Immunology 14: 585-600, (2014)), or anantigen-binding fragments thereof comprising one, two, three, four, fiveand/or six of their respective CDR sequences.

The IL-23 antagonists of the present disclosure can also be used incombination with one or more antagonists of other cytokines (e.g.antibodies), including but not limited to, IL17A, IL17F, TNF-α, IL-1β,IL-6 and TGF-β. See, e.g., Veldhoen, Immunity 24:179-189 (2006); Dong,Nat. Rev. Immunol. 6(4):329-333 (2006).

In various aspects, the IL-23 antagonists disclosed herein compriseantigen binding fragments of antibodies, such as fragments of any of theIL-23 antagonist antibodies referred to herein. Such fragments include,but are not limited to Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)₂,nanobodies, and diabodies.

The term “IL23 antagonist” also encompasses antagonists such as aptamers(see, e.g., U.S. Pat. Appl. Pub. No. 2007066550), peptides (e.g., asdisclosed in Quiniou et al., Am J Physiol Regul Integr Comp Physiol.2014 Nov. 15; 307(10):R1216-30), orally stable peptides against theIL-23 receptor, or small molecule inhibitors (e.g., SyntaPharmaceuticals' STA-5326).

V. CCL20 Levels for Diagnosis, Treatment, and Monitoring ofIL23-Mediated Diseases

IL23 pathway biomarkers that are differentially expressed in subjectshaving an IL23-mediated disease such as CD, for example CCL20, can beapplied to predicting clinical outcomes when the subjects are treatedwith a certain therapy, for example, an IL23 antagonist (e.g., ananti-IL23 antibody targeting, e.g., the p19 subunit of IL23, such asMEDI2070, or an antigen-binding fragment thereof). In some aspects, asingle IL23 pathway biomarker, e.g., CCL20, can be used. In otheraspects, CCL20 can be combined with other IL23 pathway biomarkers, suchas IL22 and/or LCN2. In other aspects, additional biomarkers, e.g.,biomarkers indicative of inflammation such as FCP and/or CRP, can becombined with CCL20.

In some aspects, the IL23 antagonist is an anti-IL23 antibody orantigen-binding fragment thereof which can specifically bind to the p19subunit of IL23 (SEQ ID NO: 15), to the p40 subunit of IL23 (SEQ ID NO:16), or both, for example the anti-IL23 antibody MEDI2070, which targetsthe p19 subunit of IL23.

In some aspects, the anti-IL23 antibody or antigen-binding fragmentthereof comprises the heavy chain (HC) (SEQ ID NO: 17) and/or the lightchain (SEQ ID NO: 18) of MEDI2070, or an antigen-binding fragment,variant, or derivative thereof. In some aspects, the anti-IL23 antibodyor antigen-binding fragment thereof comprises the heavy chain variableregion (VH) (SEQ ID NO: 7) and/or the light chain variable region (VL)(SEQ ID NO: 8) of MEDI2070. In some aspects, the anti-IL23 antibody orantigen-binding fragment thereof comprises at least one of thecomplementarity determining regions of MEDI2070 (SEQ ID NOS: 33-38). Insome aspects, the anti-IL23 antibody or antigen-binding fragment thereofcomprises a VH region comprising the sequence of SEQ ID NO: 45 and/or aVL region comprising the sequence of SEQ ID NO: 46, or anantigen-binding fragment, variant, or derivative thereof. In someaspects, the anti-IL23 antibody or antigen-binding fragment thereofcomprises at least one of the complementarity determining regions of SEQID NOS: 47-49 (CDRs of the VH of SEQ ID NO: 45) and/or SEQ ID NOS: 50-52(CDRs of the VL of SEQ ID NO:46).

In other aspects, the IL23 antagonist is an anti-IL23 antibody selectedfrom ustekinumab or briakinumab (targeting the p40 subunit of IL23),guselkumab, tildrakizumab, BI-655066 or LY-3074828 (targeting the p19subunit of IL23), an antigen-binding fragment thereof, or a combinationthereof. In other aspects, the IL23 antagonist is a molecule (e.g., anantibody) competing for binding to IL23 with an antibody selected fromustekinumab or briakinumab (targeting the p40 subunit of IL23),guselkumab, tildrakizumab, BI-655066 or LY-3074828 (targeting the p19subunit of IL23), an antigen-binding fragment thereof, or a combinationthereof.

This finding can be applied, for example, to devise new methods ofdetermining treatment (e.g., by selecting patients as candidates for acertain therapy), methods of treating an IL-23-mediated disease, methodsof monitoring efficacy of therapeutic agents (e.g., anti-IL23antibodies) to treat IL23-mediated diseases and disorders, or methods toadjust formulations, dosage regimens, or routes of administration.

The methods disclosed herein include prescribing, initiating, and/oraltering prophylaxis and/or treatment, e.g., for an IL23-mediateddisease such as CD, based at least in part on a subject's expressionlevel of CCL20, alone or in combination with one or more additionalbiomarkers. In a particular aspect, such additional biomarker is an IL23pathway biomarker such as IL22 and/or LCN2. In another aspect, suchadditional biomarker is an inflammation biomarker such as CRP and/orFCP.

The present disclosure provides a method of determining whether to treata patient having an IL23-mediated disease or disorder with a therapeuticregimen comprising the administration of an IL23 antagonist (e.g., ananti-IL23 antibody targeting, e.g., the p19 subunit of IL23, such asMEDI2070, or an antigen-binding fragment thereof) wherein the methodcomprises: (a) measuring or instructing a clinical laboratory to measurethe level of CCL20 (and optionally levels of additional biomarkers suchas IL22, LCN2, CRP, FCP, or combinations thereof) in a sample taken fromthe patient, and (b) treating or instructing a healthcare provider totreat the patient, or suspending the treatment, not initiating thetreatment, denying the treatment, or instructing a healthcare providerto suspend, not initiate, or deny the treatment with a therapeuticregimen comprising the administration of an IL23 antagonist (e.g., ananti-IL23 antibody targeting, e.g., the p19 subunit of IL23, such asMEDI2070, or an antigen-binding fragment thereof) if the patient isdetermined to have a higher or lower level of CCL20 (and optionallylevels of additional biomarkers such as IL22, LCN2, CRP, FCP, orcombinations thereof) in the sample compared to a biomarkerpredetermined threshold level or levels, or compared to a biomarkerlevel or levels in one or more control samples.

In one aspect, the disclosure provides a method of determining whetherto treat a patient having an IL23-mediated disease or disorder with atherapeutic regimen comprising the administration of an IL23 antagonist(e.g., an anti-IL23 antibody targeting, e.g., the p19 subunit of IL23,such as MEDI2070, or an antigen-binding fragment thereof) wherein themethod comprises: (a) measuring or instructing a clinical laboratory tomeasure the level of CCL20 (and optionally levels of additionalbiomarkers such as IL22, LCN2, CRP, FCP, or combinations thereof) in asample taken from the patient, and (b) treating or instructing ahealthcare provider to treat the patient with a therapeutic regimencomprising the administration of an antibody or antigen-binding fragmentthereof that specifically binds to IL23 if the patient is determined tohave a lower or decreased level of CCL20, and higher or increased levelsof at least one optional additional biomarker such as IL22, LCN2, CRP,FCP, or a combination thereof in the sample compared to a predeterminedbiomarker threshold level or levels, or compared to a biomarker level orlevels in one or more control samples.

In one aspect, the disclosure provides a method of determining whetherto treat a patient having an IL23-mediated disease or disorder with atherapeutic regimen comprising the administration of an IL23 antagonist(e.g., an anti-IL23 antibody targeting, e.g., the p19 subunit of IL23,such as MEDI2070, or an antigen-binding fragment thereof) wherein themethod comprises (a) measuring or instructing a clinical laboratory tomeasure the level of CCL20 (and optionally levels of additionalbiomarkers such as IL22, LCN2, CRP, FCP, or combinations thereof) in asample taken from the patient, and (b) suspending the treatment, notinitiating treatment, denying the treatment, or instructing a healthcareprovider to suspend, not initiate, or deny the treatment of the patientwith a therapeutic regimen comprising the administration of an antibodyor antigen-binding fragment thereof that specifically binds to IL23 tothe patient if the patient is determined to have a higher or increasedlevel of CCL20, and lower or decreased levels of at least one optionaladditional biomarker such as IL22, LCN2, CRP, FCP, or a combinationthereof in the sample compared to a predetermined biomarker thresholdlevel or levels, or compared to a biomarker level or levels in one ormore control samples.

Also provided is a method of selecting a patient diagnosed with anIL23-mediated disease or disorder as a candidate for treatment with anIL23 antagonist (e.g., an anti-IL23 antibody targeting, e.g., the p19subunit of IL23, such as MEDI2070, or an antigen-binding fragmentthereof), comprising (a) measuring or instructing a clinical laboratoryto measure the level of CCL20 (and optionally levels of additionalbiomarkers such as IL22, LCN2, CRP, FCP, or combinations thereof) in asample taken from the patient, and (b) treating or instructing ahealthcare provider to treat the patient with an IL23 antagonist (e.g.,an anti-IL23 antibody targeting, e.g., the p19 subunit of IL23, such asMEDI2070, or an antigen-binding fragment thereof) if the patient isdetermined to have a lower or decreased level of CCL20, and higher orincreased levels of at least one optional additional biomarker such asIL22, LCN2, CRP, FCP, or a combination thereof in the sample compared toa predetermined threshold level or levels, or compared to a biomarkerlevel or levels in one or more control samples.

Also provided is method of selecting a patient diagnosed with anIL23-mediated disease or disorder as a candidate for treatment with anIL23 antagonist (e.g., an anti-IL23 antibody targeting, e.g., the p19subunit of IL23, such as MEDI2070, or an antigen-binding fragmentthereof), comprising (a) measuring or instructing a clinical laboratoryto measure the level of CCL20 (and optionally levels of additionalbiomarkers such as IL22, LCN2, CRP, FCP, or combinations thereof) in asample taken from the patient, and (b) suspending the treatment, notinitiating treatment, denying the treatment, or instructing a healthcareprovider to suspend, not initiate, or deny the treatment of the patientwith an IL23 antagonist (e.g., an anti-IL23 antibody targeting, e.g.,the p19 subunit of IL23, such as MEDI2070, or an antigen-bindingfragment thereof) to the patient if the patient is determined to have ahigher or increased level of CCL20, and lower or decreased levels of atleast one optional additional biomarker such as IL22, LCN2, CRP, FCP, ora combination in the sample compared to a predetermined threshold levelor levels, or compared to a biomarker level or levels in one or morecontrol samples.

In some aspects, the methods disclosed can entail ordering and/orperforming one or more additional assays. For example, if the level ofCCL20 (and optionally levels of additional biomarkers such as IL22,LCN2, CRP, FCP, or combinations thereof) (e.g., a protein expressionlevel or a gene expression level) is determined to be within a normalrange (i.e., not elevated), the CCL20 assay (or assay for anotherbiomarker) may be repeated to rule out a false negative result, and/orone or more additional CCL20 assays (or other biomarker's assays) may beperformed to monitor the subject's status. Conversely, if the CCL20level (e.g., a protein expression level or a gene expression level) isdetermined to be elevated, it may be desirable repeat the CCL20 assay torule out a false positive result.

In some aspects, the predetermined CCL20 threshold level is at leastabout 22.6 pg/mL as measured in serum using a CCL20 immunoassay(including, e.g., the CCL20 immunoassay described in Example 3). In someaspects, the predetermined IL22 threshold level is at least about 15.6pg/mL as measured in serum using an IL22 immunoassay (including, e.g.,the IL22 immunoassay described in Example 4); and/or the predeterminedLCN2 threshold level is at least about 215 ng/mL as measured in serumusing an LCN2 immunoassay (including, e.g., the LCN2 immunoassaydescribed in Example 4).

In some aspects, the presence of CCL20 above or below a predeterminedthreshold level in a patient with an IL23-mediated disease can be usedin combination with one or more of biomarkers specific for such disease.For example, for patients with inflammatory bowel disease (IBD)(including, e.g., CD or UC), the measurement of CCL20 can be combinedwith measurements of other IL23 pathway biomarker levels (e.g., IL22and/or LCN2 levels) and other biomarkers such as C-reactive protein(CRP) and/or fetal calprotectin (FCP) levels. Accordingly, in oneaspects, levels of CCL20 can be combined with IL22, LCN2, CRP and/or FCPlevels in any of the methods disclosed herein (i) to determine whether apatient suffering an IL23-mediated disease (e.g., IBD, CD or UC) iseligible or non-eligible for a specific treatment or will respond to aspecific treatment with an IL23 antagonist (e.g., an anti-IL23 antibodytargeting, e.g., the p19 subunit of IL23, such as MEDI2070, or anantigen-binding fragment thereof), (ii) to determine whether a specifictreatment (e.g., with an IL23 antagonist such as MEDI2070) shouldcommence, be suspended, or be modified, (iii) to diagnose whether thedisease (e.g., IBD, CD or UC) is treatable or not treatable with aspecific therapeutic agent, or (iv) to prognosticate or predict theoutcome of treatment of the disease (e.g., IBD, CD or UC) with aspecific therapeutic agent, etc. if CRP and/calprotectin levels arehigh.

In some aspects, mean levels of C-reactive protein are high or elevated(and thus indicative of having active disease) if they are ≥5 mg/L asmeasured using an assay suitable for measuring CRP levels in a patient,including, e.g., the Dade Behring hs-CRP immunoturbidometric assayfollowing the manufacturer's instructions. In some aspects, mean levelsof fecal calprotectin are high or elevated (and thus indicative ofhaving active disease) if they are ≥250 μg/g as measured using an assaysuitable for measuring fecal calprotectin levels in a patient,including, e.g., Phadia ELIA™ Calprotectin assay following themanufacturer's instructions. In other aspects, mean levels of C-reactiveprotein are high or elevated if they are at least about 2, 3, 4, 5, 6,7, 8, 9 or 10 mg/L as measured using an assay suitable for measuring CRPlevels in a patient, including, e.g., the Dade Behring hs-CRPimmunoturbidometric assay following the manufacturer's instructions. Insome aspects, mean levels of fecal calprotectin are high or elevated ifthey are at least 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 310, 320, 330, 340 or 350 μg/g as measuredusing an assay suitable for measuring calprotectin levels in a patient,including, e.g., the Phadia ELIA™ Calprotectin assay following themanufacturer's instructions. In some aspects, the levels of fecalcalprotectin are high or elevated if they are ≥250 μg/g, ≥200 μg/g, ≥150μg/g, ≥100 μg/g, or at least about 100 μg/g to at least about 250 μg/gas measured using an assay suitable for measuring calprotectin levels ina patient, including, e.g., the Phadia ELIA™ Calprotectin assayfollowing the manufacturer's instructions. A person skilled in the artwould appreciate that different methods to quantify C-reactive proteinor calprotectin can be applied in the methods disclosed herein. See,e.g., U.S. Pat. No. 8,541,180; U.S. Pat. Appl. Publ. Nos. US20140227725or US20140227725; and PCT Publ. Nos. WO2012175616, WO2010062663,WO2012175602, WO2013132338, or WO2013132347, which are hereinincorporated by reference in their entireties.

A person skilled in the art would understand that CCL20 levels (e.g., aprotein expression level or a gene expression level) can be usedaccording to the methods disclosed herein, including but not limited totreatment, diagnostic, and monitoring methods, as positive selectors,i.e., a specific action would be taken (e.g., treating a patient havingan IL23-mediated disease with an IL23 antagonist) if the CCL20 levels(e.g., a protein expression level or a gene expression level) in asample taken from the patient are below a predetermined CCL20 thresholdlevel, or are decreased relative to the CCL20 levels in one or morecontrol samples.

A person skilled in the art would also understand that CCL20 levels(e.g., a protein expression level or a gene expression level) can beused according to the methods disclosed herein, including but notlimited to treatment, diagnostic, and monitoring methods, as negativeselectors, i.e., a specific action would not be taken (e.g., treating apatient having an IL23-mediated disease with an IL23 antagonist) if theCCL20 levels (e.g., a protein expression level or a gene expressionlevel) in a sample taken from the patient are above a predeterminedCCL20 threshold level, or are increased relative to the CCL20 levels inone or more control samples.

In one aspect, the disclosure includes methods, assays, and kits tofacilitate a determination by a healthcare provider, a healthcarebenefits provider, or a clinical laboratory to as to whether a patientwill benefit from treatment with an IL23 antagonist (e.g., an anti-IL23antibody targeting, e.g., the p19 subunit of IL23, such as MEDI2070, oran antigen-binding fragment thereof).

The methods assays and kits provided herein also facilitate adetermination by a healthcare provider, a healthcare benefits provider,or a clinical laboratory to as to whether a patient will benefit fromtreatment with any other IL23 antagonist (e.g., an anti-IL23 antibodytargeting, e.g., the p19 subunit of IL23, such as MEDI2070, or anantigen-binding fragment thereof) disclosed herein or known to those ofordinary skill in the art.

In one aspect, the methods disclosed herein include making a diagnosis,which may be a differential diagnosis, based at least in part on thelevels of CCL20 of a patient. In some aspects, the methods disclosedherein include informing the subject of a result of the CCL20 assayand/or of a diagnosis based at least in part on the CCL20 level. Thepatient can be informed verbally, in writing, and/or electronically.

This diagnosis can also be recorded in a patient medical record. Forexample, in various aspects, the diagnosis of an IL23-mediated disease(e.g., CD) treatable with a specific IL23 antagonist (e.g., an anti-IL23antibody targeting, e.g., the p19 subunit of IL23, such as MEDI2070, oran antigen-binding fragment thereof) is recorded in a medical record.The term “medical record” or “patient medical record” refers to anaccount of a patient's examination and/or treatment that typicallyincludes one or more of the following: the patient's medical history andcomplaints, the physician's physical findings, the results of diagnostictests and procedures, and patient medications and therapeuticprocedures. A medical record is typically made by one or more physiciansand/or physicians' assistants and it is a written, transcribed orotherwise recorded record and/or history of various illnesses orinjuries requiring medical care, and/or inoculations, and/or allergies,and/or treatments, and/or prognosis, and/or frequently healthinformation about parents, siblings, and/or occupation. The record maybe reviewed by a physician in diagnosing the condition.

The medical record can be in paper form and/or can be maintained in acomputer-readable medium. The medical record can be maintained by alaboratory, physician's office, a hospital, a healthcare maintenanceorganization, an insurance company, and/or a personal medical recordwebsite. In some aspects, a diagnosis, based at least in part on theCCL20 level, is recorded on or in a medical alert article such as acard, a worn article, and/or a radiofrequency identification (RFID) tag.As used herein, the term “worn article” refers to any article that canbe worn on a subject's body, including, but not limited to, a tag,bracelet, necklace, arm band, or head band.

As used herein, the term “diagnosis” means detecting a disease ordetermining the stage or degree of a disease. Usually, a diagnosis of adisease is based on the evaluation of one or more factors and/orsymptoms that are indicative of the disease. That is, a diagnosis can bemade based on the presence, absence or amount of a factor which isindicative of presence or absence of the disease or disorder. Eachfactor or symptom that is considered to be indicative for the diagnosisof a particular disease does not need be exclusively related to theparticular disease, e.g. there may be differential diagnoses that can beinferred from a diagnostic factor or symptom. Likewise, there may beinstances where a factor or symptom that is indicative of a particulardisease is present in an individual that does not have the particulardisease.

The term “diagnosis” also encompasses determining the therapeutic effectof a drug therapy, or predicting the pattern of response to a drugtherapy. The diagnostic methods may be used independently, or incombination with other diagnosing and/or staging methods known in themedical arts for a particular disease.

As used herein, the term “differential diagnosis” refers to thedetermination of which of two or more diseases with similar symptoms islikely responsible for a subject's symptom(s), based on an analysis ofthe clinical data. The term is also used to refer to the determinationof whether a patient is susceptible to treatment with an IL23 antagonist(e.g., an anti-IL23 antibody targeting, e.g., the p19 subunit of IL23,such as MEDI2070, or an antigen-binding fragment thereof) depending onwhether the measured CCL20 levels in a patient sample are below apredetermined threshold level, or decreased relative to the level in oneor more control samples.

The term “prognosis” as used herein refers to a prediction of theprobable course and outcome of a clinical condition or disease. Aprognosis is usually made by evaluating factors or symptoms of a diseasethat are indicative of a favorable or unfavorable course or outcome ofthe disease. The phrase “determining the prognosis” as used hereinrefers to the process by which the skilled artisan can predict thecourse or outcome of a condition in a patient. The term “prognosis” doesnot refer to the ability to predict the course or outcome of a conditionwith 100% accuracy. Instead, the skilled artisan will understand thatthe term “prognosis” refers to an increased probability that a certaincourse or outcome will occur; that is, that a course or outcome is morelikely to occur in a patient exhibiting a given condition, when comparedto those individuals not exhibiting the condition.

The terms “favorable prognosis” and “positive prognosis,” or“unfavorable prognosis” and “negative prognosis” as used herein arerelative terms for the prediction of the probable course and/or likelyoutcome of a condition or a disease. A favorable or positive prognosispredicts a better outcome for a condition than an unfavorable ornegative prognosis. In a general sense, a “favorable prognosis” is anoutcome that is relatively better than many other possible prognosesthat could be associated with a particular condition, whereas anunfavorable prognosis predicts an outcome that is relatively worse thanmany other possible prognoses that could be associated with a particularcondition. Typical examples of a favorable or positive prognosis includea better than average remission rate, a lower propensity for metastasis,a longer than expected life expectancy, differentiation of a benignprocess from a cancerous process, and the like.

The disclosure includes methods of treating an IL23-mediated mediateddisease in a subject based on the changes in expression of IL23 pathwaybiomarkers, for example, CCL20. The disclosure provides a method oftreating a patient having an IL23-mediated disease or disorder,comprising: administering an IL23 antagonist (e.g., an anti-IL23antibody targeting, e.g., the p19 subunit of IL23, such as MEDI2070, oran antigen-binding fragment thereof) to the patient if the patient isdetermined to have a lower or decreased level of CCL20 in one or moresamples taken from the patient compared to a predetermined CCL20threshold level, or compared to the CCL20 level in one or more controlsamples. In some aspects, a sample is obtained from a patient and issubmitted for measurement of the level of CCL20 in the sample.

The disclosure also provides a method of treating a patient having anIL23-mediated disease or disorder comprising: (a) submitting a sampletaken from the patient for measurement of the levels of CCL20 in thesample, and (b) administering an antibody or antigen-binding fragmentthereof that specifically binds to IL23 (e.g., to the p19 subunit ofIL23, such as MEDI2070) to the patient if the patients has a lower ordecreased level of CCL20 in the samples taken compared to apredetermined CCL20 threshold level, or compared to the CCL20 level inone or more control samples.

Also provided is method of treating a patient having an IL23-mediateddisease or disorder comprising: (a) submitting a sample taken from thepatient for measurement of the levels of CCL20 in the sample, and (b)suspending or not initiating the administration of an antibody orantigen-binding fragment thereof that specifically binds to IL23 (e.g.,to the p19 subunit of IL23, such as MEDI2070) to the patient if thepatients has a higher or increased level of CCL20 in the sample comparedto a predetermined CCL20 threshold level, or compared to the CCL20 levelin one or more control samples.

The disclosure also provides a method of treating a patient having anIL23-mediated disease or disorder comprising: (a) measuring the levelsof CCL20 in a sample obtained from the patient; (b) determining thelevel of CCL20 in the sample, and, (c) advising a healthcare provider toadminister an antibody or antigen-binding fragment thereof thatspecifically binds to IL23 (e.g., to the p19 subunit of IL23, such asMEDI2070) to the patient if the patient is determined to have a lower ordecreased level of CCL20 in the sample compared to a predetermined CCL20threshold level, or compared to the CCL20 levels in one or more controlsamples; or to suspend or deny the administration of an antibody orantigen-binding fragment thereof that specifically binds to IL23 to thepatient if the patient is determined to have a higher or increased levelof CCL20 in the sample compared to a predetermined CCL20 thresholdlevel, or compared to the CCL20 level in one or more control samples.

Also provided is a method of treating a patient having an IL23-mediateddisease or disorder comprising: (a) submitting a sample taken from apatient for measurement of the level of CCL20 in a sample obtained fromthe patient, and (b) administering an antibody or antigen-bindingfragment thereof that specifically binds to IL23 (e.g., to the p19subunit of IL23, such as MEDI2070) to the patient if the patient isdetermined to have a lower or decreased level of CCL20 in the samplecompared to a predetermined CCL20 threshold level, or compared to theCCL20 level in one or more control samples; or suspending, notinitiating, or denying the administration of an antibody orantigen-binding fragment thereof that specifically binds to IL23 (e.g.,to the p19 subunit of IL23, such as MEDI2070) to the patient if thepatient is determined to have a higher or increased level of CCL20 inthe sample compared to a predetermined CCL20 threshold level, orcompared to the CCL20 level in one or more control samples.

In some aspects, the method comprises administering to the subject aneffective amount of an IL23 antagonist (e.g., an anti-IL23 antibodytargeting, e.g., the p19 subunit of IL23, such as MEDI2070, or anantigen-binding fragment thereof). In some aspects, the IL23-mediateddisease is a pulmonary disease, an inflammatory bowel disease, a chronicinflammatory skin disease, an inflammatory disease, an autoimmunedisease, a neurodegenerative disease, an infection, or a cancer. In someaspects, the IL23-mediated disease or disorder is selected from thegroup consisting of: asthma, IPF, COPD. Crohn's disease, ulcerativecolitis (UC), celiac disease, atopic dermatitis, allergic contactdermatitis, eczema, psoriasis, alopecia areata, palmoplantar pustulosis,psoriatic arthritis, anklyosing spondylitis, arthritis, rheumatoidarthritis (RA), a rheumatic disorder, ANCA vasculitis, Bechet's disease,autoimmune thyroiditis, type 1 diabetes, multiple sclerosis (MS),Sjogren's syndrome (SS), systemic lupus erythematosus (SLE), Alzheimer'sdisease, mycobacterial disease, leishmaniasis, a fungal infection, aviral infection, gastric cancer, colorectal cancer, esophageal cancer,leukemia, hepatitis B virus (HBV)-related hepatocellular carcinoma,breast cancer, lung cancer, and nasopharyngeal cancer.

In some aspects, the patient's CCL20 level is measured in an immunoassayas described herein employing one or more anti-CCL20 antibodies orantigen binding fragments thereof which recognize human CCL20 orantigen-binding fragments, variants or derivatives thereof. In someaspects, the sample is obtained from the patient and is submitted formeasurement of the level of CCL20 in the sample, for example, to aclinical laboratory.

In some aspects of the above treatment methods, the patient's CCL20level (e.g., DNA or RNA level) are measured in an assay employing one ormore oligonucleotide probes capable of specifically measuring theexpression levels of the CCL20 gene.

In some aspects, the CCL20 detection assay (e.g., an immunoassay) isperformed on a sample obtained from the patient, by the healthcareprofessional treating the patient (e.g., using an immunoassay asdescribed herein including, e.g., the immunoassay described in Example3, formulated as a “point of care” diagnostic kit). In some aspects, asample is obtained from the patient and is submitted, e.g., to aclinical laboratory, for measurement of the CCL20 level in the sampleaccording to the healthcare professional's instructions (e.g., using animmunoassay as described herein including, e.g., the immunoassaydescribed in Example 3).

In some aspects, the clinical laboratory performing the assay willadvise the healthcare provide as to whether the patient can benefit fromtreatment with an IL23 antagonist (e.g., an anti-IL23 antibodytargeting, e.g., the p19 subunit of IL23, such as MEDI2070, or anantigen-binding fragment thereof) based on whether the patient's CCL20level is below a predetermined CCL20 threshold value or is decreasedrelative to one or more control samples.

In some aspects, the second measurement is conducted 1, 2, 4, 8, 12, or28 weeks, or at intervening times, after administering the IL23 antibodyor antigen-binding fragment thereof that specifically binds to IL23(e.g., to the p19 subunit of IL23, such as MEDI2070).

In some aspects, this disclosure includes a method of treating a patienthaving an IL23-mediated disease over a period of time, comprising:measuring a first CCL20 level (e.g., protein expression level or geneexpression level) in a first sample taken from the patient, orsubmitting a first sample taken from the patient for measurement of afirst CCL20 level in the sample, wherein the patient's CCL20 level is,for example, measured in an immunoassay, including, e.g., an immunoassaydescribed in Example 3, employing one or more anti-CCL20 antibodies orantigen binding fragments thereof which recognize human CCL20, andadministering an IL23 antagonist (e.g., an anti-IL23 antibody targeting,e.g., the p19 subunit of IL23, such as MEDI2070, or an antigen-bindingfragment thereof) to the patient if the patient's CCL20 level in thefirst sample is below a predetermined CCL20 threshold level, or islowered relative to the CCL20 level in one or more control samples. Thetest can be performed by a healthcare provider or a clinical laboratoryas noted above.

According to these aspects, the method can further comprise: measuring asecond CCL20 level (e.g., protein expression level or gene expressionlevel) in a second sample taken from the patient, or submitting a secondsample taken from the patient for measurement of a second CCL20 level inthe sample, wherein the patient's CCL20 level is again measured, forexample, in an immunoassay, including, e.g., an immunoassay described inExample 3, employing one or more anti-CCL20 antibodies or antigenbinding fragments thereof which recognize human CCL20; comparing thefirst and second CCL20 level in the patient, and altering the dose,e.g., increasing or maintaining the amount or frequency of the IL23antagonist administered to the patient (e.g., an anti-IL23 antibodytargeting, e.g., the p19 subunit of IL23, such as MEDI2070, or anantigen-binding fragment thereof). In some aspects, IL23 antagonisttherapy can be discontinued if, e.g., the patient's CCL20 level in thesecond sample is lower than the CCL20 level in the first sample.

In some aspects, the amount or frequency of the IL23 antagonistadministered to the patient (e.g., an anti-IL23 antibody targeting,e.g., the p19 subunit of IL23, such as MEDI2070, or an antigen-bindingfragment thereof) can be maintained or reduced if the patient's CCL20level in the second sample is higher than or about the same as the CCL20level in the first sample.

In certain aspects, in all the treatment methods disclosed herein, a“loading” dose of an IL23 antagonist (e.g., an anti-IL23 antibodytargeting, e.g., the p19 subunit of IL23, such as MEDI2070, or anantigen-binding fragment thereof) is administered to achieve a desiredtherapeutic level in the patient. If the loading dose does not affectthe patient's CCL20 level (e.g., protein expression levels or geneexpression levels) significantly or the patient's CCL20 level decreases,a decision could be made to discontinue treatment—e.g., to use anon-IL23 antagonist therapy.

If the loading dose results in an increased CCL20 level in the patient adecision could be made to reduce the dose size or frequency to a“maintenance” dose. It is important to note that the methods providedhere are guidelines for a healthcare provider to administer treatment,and the ultimate treatment decision will be based on the healthcareprovider's sound judgment.

In some aspects, results of an immunoassay as provided herein can besubmitted to a healthcare benefits provider for determination of whetherthe patient's insurance will cover treatment with an IL23 antagonist(e.g., an anti-IL23 antibody targeting, e.g., the p19 subunit of IL23,such as MEDI2070, or an antigen-binding fragment thereof).

In some aspects, this disclosure includes a method of treating a patienthaving an IL23-mediated disease comprising: measuring, e.g., in aclinical laboratory, the CCL20 level (e.g., protein expression level orgene expression level) in a first sample obtained from a patient havingan IL23-mediated disease, e.g., a sample provided by a healthcareprovider, wherein the patient's CCL20 level in the first sample is, forexample, measured in an immunoassay, including, e.g., an immunoassaydescribed in Example 3, employing one or more anti-CCL20 antibodies orantigen binding fragments thereof which recognize human CCL20,determining whether the patient's CCL20 level in the first sample isbelow a predetermined CCL20 level, or is decreased relative to the CCL20level in one or more control samples; and advising a healthcare providerto administer an IL23 antagonist (e.g., an anti-IL23 antibody targeting,e.g., the p19 subunit of IL23, such as MEDI2070, or an antigen-bindingfragment thereof) to the patient if the patient's CCL20 level is below apredetermined CCL20 threshold level, or are decreased relative to theCCL20 level in one or more control samples.

In some aspects, these methods can further comprise: measuring the CCL20level (e.g., protein expression level or gene expression level) in asecond sample obtained from the patient, e.g., a sample provided by ahealthcare provider, wherein the patient's CCL20 level is againmeasured, for example, in an immunoassay, including, e.g., animmunoassay described in Example 3, employing one or more anti-CCL20antibodies or antigen binding fragments thereof which recognize humanCCL20; determining whether the patient's CCL20 level in the secondsample is higher than, about the same as, or lower than the CCL20 levelmeasured in the first sample; and advising a healthcare provider toadjust the IL23 antagonist therapy (e.g., a therapy comprising theadministration of an anti-IL23 antibody targeting, e.g., the p19 subunitof IL23, such as MEDI2070, or an antigen-binding fragment thereof) ifindicated, e.g., to increase or maintain the amount or frequency of theIL23 antagonist administered to the patient, or discontinuing IL23antagonist therapy, if the patient's CCL20 level in the second sample islower than the CCL20 level in the first sample, or to maintain or reducethe amount or frequency of the IL23 antagonist administered to thepatient if the patient's CCL20 level in the second sample is higher thanor about the same as the CCL20 level in the first sample.

In some aspects, a sample is obtained from the patient and is submitted,e.g., to a clinical laboratory, for measurement of the level of CCL20alone or in combination with the level of at least another IL23 pathwaybiomarker, e.g., IL22 and/or LCN2; at least one inflammation biomarker,e.g., CRP and/or FCP; or a combination thereof (e.g., protein expressionlevel or gene expression level) in the sample, e.g., using animmunoassay.

In some aspects, the clinical laboratory performing the assay willadvise the healthcare provide as to whether the patient can benefit fromtreatment with an IL23 antagonist (e.g., an anti-IL23 antibodytargeting, e.g., the p19 subunit of IL23, such as MEDI2070, or anantigen-binding fragment thereof) based on whether the patient's CCL20level (e.g., protein expression level or gene expression level) is belowa predetermined CCL20 threshold value or is low relative to one or morecontrol samples.

In some aspects, methods of treatment contemplated herein (e.g., for anIL23-mediated disease such Crohn's disease) comprise administering tothe subject an antibody or antigen-binding fragment thereof targetingIL23 (e.g., to the p19 subunit of IL23, such as MEDI2070) in asufficient amount and/or at sufficient interval to achieve and/ormaintain a certain quantity of IL23-specific antibody per volume ofserum, using, for example, an assay as described herein.

For example, the antibody or antigen-binding fragment thereof targetingIL23 (e.g., to the p19 subunit of IL23, such as MEDI2070) can be givento achieve at least about 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35ng/ml, 40 ng/ml, 45 ng/ml, 50 ng/ml, 55 ng/ml, 60 ng/ml, 65 ng/ml, 70ng/ml, 75 ng/ml, 80 ng/ml, 85 ng/ml, 90 ng/ml, 95 ng/ml, 100 ng/ml, 120ng/ml, 140 ng/ml, 160 ng/ml, 180 ng/ml, 200 ng/ml, 220 ng/ml, 240 ng/ml,260 ng/ml, 280 ng/ml, 300 ng/ml, 320 ng/ml, 340 ng/ml, 360 ng/ml, 380ng/ml, 400 ng/ml, 420 ng/ml, 440 ng/ml, 460 ng/ml, 480 ng/ml, 500 ng/ml,520 ng/ml, 540 ng/ml, 560 ng/ml, 580 ng/ml, 600 ng/ml, 620 ng/ml, 640ng/ml, 660 ng/ml, 680 ng/ml, 700 ng/ml, 720 ng/ml, 740 ng/ml, 760 ng/ml,780 ng/ml, 800 ng/ml, 820 ng/ml, 840 ng/ml, 860 ng/ml, 880 ng/ml, 900ng/ml, 920 ng/ml, 940 ng/ml, 960 ng/ml, 980 ng/ml, or 1000 ng/ml inserum.

In a further embodiment, the antibody or antigen-binding fragmentthereof targeting IL23 (e.g., to the p19 subunit of IL23, such asMEDI2070) can be given to achieve a concentration of IL23-specificantibody in serum from about 12.5 ng/ml to about 1000 ng/ml. Those ofskill in the art will understand that the amounts given here apply to afull-length antibody or immunoglobulin molecule; if an antigen bindingfragment thereof is used, the absolute quantity will differ from thatgiven in a manner that can be calculated based on the molecular weightof the fragment.

In some aspects, methods of treatment contemplated herein (e.g., for anIL23-mediated disease such as Crohn's disease) comprise administering tothe subject an IL23 antagonist (e.g., an anti-IL23 antibody targeting,e.g., the p19 subunit of IL23, such as MEDI2070, or an antigen-bindingfragment thereof) in an amount and at an interval of: 15-54 mg every0.5-1.5 months; 55-149 mg every 1.5-4.5 months; 150-299 mg every 4-8months; or 300-1100 mg every 4-12 months.

In some aspects, the amount and interval are: 15-21 mg every 0.5-1.0month; 55-70 mg every 1.5-3.0 months; 150-260 mg every 4-6 months; or300-700 mg every 4-8 months. In some aspects, the amount and intervalare: 21 mg every month; 70 mg every 3 months; 210 mg every 6 months; or700 mg every 6 months. In some aspects, the amount and interval are: 210mg every 3 months or 700 mg every 3 months. In some aspects, the amountand interval are: 210 mg every 1 month or 700 mg every 1 month.

In some aspects of the methods, the IL23 antagonist (e.g., an anti-IL23antibody targeting, e.g., the p19 subunit of IL23, such as MEDI2070, oran antigen-binding fragment thereof) is administered intravenously (IV).In some aspects of the methods, the IL23 antagonist (e.g., an anti-IL23antibody targeting, e.g., the p19 subunit of IL23, such as MEDI2070, oran antigen-binding fragment thereof) is administered subcutaneously(SC).

In some aspects, the IL23 antagonist (e.g., an anti-IL23 antibodytargeting, e.g., the p19 subunit of IL23, such as MEDI2070, or anantigen-binding fragment thereof) is administered in one or more fixeddoses. In some aspects, the doses as administered every week, every twoweeks, every three weeks, every 4 weeks, every 5 weeks, every 6 weeks,every 7 weeks, every 8 weeks, every 9 weeks every 10 weeks, or every 12weeks. In some aspects, the dose comprises about 1 mg, 2 mg, 3 mg, 4 mg,5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg,160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg,250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg,340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg,430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg, 510 mg,520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg,610 mg, 620 mg, 630 mg, 640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg,700 mg, 710 mg, 720 mg, 730 mg, 740 mg, 750 mg, 760 mg, 770 mg, 780 mg,790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg,880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg,970 mg, 980 mg, 990 mg, or 1000 mg. In some aspects, the dose is higherthan 1000 mg.

In some aspects, the dose is about 210 mg of IL23 antagonist (e.g., ananti-IL23 antibody targeting, e.g., the p19 subunit of IL23, such asMEDI2070, or an antigen-binding fragment thereof) administered SC.

In other aspects, the dose is about 700 mg of IL23 antagonist (e.g., ananti-IL23 antibody targeting, e.g., the p19 subunit of IL23, such asMEDI2070, or an antigen-binding fragment thereof) administered IV.

In some aspects, two, three, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50doses are administered.

In one specific aspect, the anti-IL23 antibody (e.g., MEDI2070) isadministered at a fixed dose of 210 mg Q4W for 26 doses. In anotherspecific aspects, the anti-IL23 antibody (e.g., MEDI2070) isadministered at a fixed dose of 700 mg IV Q4W for 12-weeks. In someaspects, the IV administration is followed by administration of theanti-IL23 antibody (e.g., MEDI2070) at a fixed dose of 210 mg SC Q4W for100-weeks.

The formulation, dosage regimen, and route of administration of atherapeutic agent, e.g., and IL23 antagonist (e.g., an anti-IL23antibody targeting, e.g., the p19 subunit of IL23, such as MEDI2070, oran antigen-binding fragment thereof), can be adjusted to provide aneffective amount for an optimum therapeutic response according to themethod disclosed herein. With regard to the administration of an IL23antagonist, the antagonist may be administered through any suitablemeans, compositions and routes known in the art. With regard to dosageregiments, a single bolus can be administered, several divided doses canbe administered over time or the dose can be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.

The IL23 antagonist (e.g., an anti-IL23 antibody targeting, e.g., thep19 subunit of IL23, such as MEDI2070, or an antigen-binding fragmentthereof) may be administered by any suitable technique, including butnot limited to, parenterally, topically, or by inhalation. If injected,the pharmaceutical composition can be administered, for example, viaintra-articular, intravenous, intramuscular, intralesional,intraperitoneal or cutaneous routes (including intra-, trans- orsub-dermal, and subcutaneous), by bolus injection, or continuousinfusion. In some aspects, the pharmaceutical composition isadministered by an intravenous route. In some aspects the pharmaceuticalcomposition is administered by a subcutaneous route.

In further aspects, the compositions are administered by oral, buccal,rectal, intratracheal, gastric, or intracranial routes. Localizedadministration, e.g. at a site of disease or injury is contemplated, forexample, by enema or suppository for conditions involving thegastrointestinal tract. Also contemplated are transdermal delivery andsustained release from implants. Delivery by inhalation includes, forexample, nasal or oral inhalation, use of a nebulizer, inhalation of theantagonist in aerosol form, and the like. Other alternatives includeeyedrops; oral preparations including pills, syrups, lozenges or chewinggum; and topical preparations such as lotions, gels, sprays, andointments, prefilled syringes and autoinjectors.

Advantageously, the IL23 antagonist (e.g., an anti-IL23 antibodytargeting, e.g., the p19 subunit of IL23, such as MEDI2070, or anantigen-binding fragment thereof) can be administered in the form of acomposition comprising one or more additional components such as aphysiologically acceptable carrier, excipient or diluent. Optionally,the composition additionally comprises one or more physiologicallyactive agents for combination therapy.

A pharmaceutical composition may comprise an IL23 antagonist (e.g., ananti-IL23 antibody targeting, e.g., the p19 subunit of IL23, such asMEDI2070, or an antigen-binding fragment thereof) together with one ormore substances selected from the group consisting of a buffer, anantioxidant such as ascorbic acid, a low molecular weight polypeptide(such as those having fewer than 10 amino acids), a protein, an aminoacid, a carbohydrate such as glucose, sucrose or dextrins, a chelatingagent such as EDTA, glutathione, a stabilizer, and an excipient.

Neutral buffered saline or saline mixed with conspecific serum albuminare examples of appropriate diluents. In accordance with appropriateindustry standards, preservatives such as benzyl alcohol may also beadded. The composition may be formulated as a lyophilizate usingappropriate excipient solutions (e.g., sucrose) as diluents.

In some aspects, the IL23 antagonist (e.g., an anti-IL23 antibodytargeting, e.g., the p19 subunit of IL23, such as MEDI2070, or anantigen-binding fragment thereof) can be provided at a concentration ofabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,165, 170, 175, 180, 185, 190, 195 or 200 mg/ml.

Exemplary formulations useful for the present invention are those thatinclude a glutamic acid, citric acid or acetic acid buffer at anappropriate pH, from 4.5 to 5.2, an excipient such as sucrose, glycine,proline, glycerol, and/or sorbitol at an appropriate concentration suchas 1 to 20% (w/v), and a surfactant such as a non-ionic surfactant likepolysorbate (polysorbate 20 or 80) or poloxamers (poloxamer 1888) at anappropriate concentration of 0.001%-0.1% (w/v). Such formulations aredisclosed in U.S. Pat. No. 6,171,586 and WIPO Published ApplicationsNos. WO20100027766 and WO2011088120. In some aspects, the formulationscomprise sodium acetate, sucrose and polysorbate 20.

In some aspects, the formulations comprise 70 mg/mL anti-IL23 antibody(e.g., MEDI2070), 10 mM sodium acetate, 9% (w/v) sucrose and 0.004%(w/v) polysorbate 20, at pH 5.2. Suitable components are nontoxic torecipients at the dosages and concentrations employed. Further examplesof components that may be employed in pharmaceutical formulations arepresented in any Remington's Pharmaceutical Sciences including the21^(st) Ed. (2005), Mack Publishing Company, Easton, Pa.

VI. Combination Treatments

Particular aspects of methods of the invention involve the use of ananti-IL23 antibody (e.g., an anti-IL23 antibody targeting, e.g., the p19subunit of IL23, such as MEDI2070, or an antigen-binding fragmentthereof) and one or more additional IL23 antagonists as described in thesection of the description entitled “IL23 Antagonists” in combinationwith additional therapeutic agents.

In some aspects, the IL23 antagonist is an anti-IL23 antibody which canspecifically bind to the p19 subunit of IL23 (SEQ ID NO: 15), to the p40subunit of IL23 (SEQ ID NO: 16), or both.

In some aspects, the anti-IL23 antibody or antigen-binding fragmentthereof comprises the heavy chain (HC) (SEQ ID NO: 17) and/or the lightchain (SEQ ID NO: 18) of MEDI2070, or an antigen-binding fragment,variant, or derivative thereof. In some aspects, the anti-IL23 antibodyor antigen-binding fragment thereof comprises the heavy chain variableregion (VH) (SEQ ID NO: 7) and/or the light chain variable region (VL)(SEQ ID NO: 8) of MEDI2070. In some aspects, the anti-IL23 antibody orantigen-binding fragment thereof comprises at least one of thecomplementarity determining regions of MEDI2070 (SEQ ID NOS: 33-38).

In some aspects, the anti-IL23 antibody or antigen-binding fragmentthereof comprises a VH region comprising the sequence of SEQ ID NO: 45and/or a VL region comprising the sequence of SEQ ID NO: 46, or anantigen-binding fragment, variant, or derivative thereof. In someaspects, the anti-IL23 antibody or antigen-binding fragment thereofcomprises at least one of the complementarity determining regions of SEQID NOS: 47-49 (CDRs of the VH of SEQ ID NO: 45) and/or SEQ ID NOS: 50-52(CDRs of the VL of SEQ ID NO: 46).

In other aspects, the IL23 antagonist is an anti-IL23 antibody selectedfrom ustekinumab or briakinumab (targeting the p40 subunit of IL23),guselkumab, tildrakizumab, BI-655066 or LY-3074828 (targeting the p19subunit of IL23), an antigen-binding fragment thereof, or a combinationthereof. In other aspects, the IL23 antagonist is a molecule (e.g., anantibody) that competes for binding to IL23 with ustekinumab orbriakinumab (targeting the p40 subunit of IL23), guselkumab,tildrakizumab, BI-655066 or LY-3074828 (targeting the p19 subunit ofIL23), an antigen-binding fragment thereof, or a combination thereof.

Examples include using combinations of an anti-IL23 antibody and one ormore other therapeutic moiety having anti-inflammatory properties (forexample, non-steroidal anti-inflammatory agents, steroids, and/orimmunomodulators), or of an anti-IL23 antibody and one or more othertreatments (e.g., surgery, ultrasound, or treatment effective to reduceinflammation). Useful agents that may be combined with an anti-IL23antibody include those used to treat, for example, Crohn's disease orulcerative colitis, such as aminosalicylate (for example, mesalamine orsubstances that are metabolized to mesalamine, including, for example,ASACOL®, SALOFALK®, PENTASA®, DIPENTUM®, COLAZIDE®, LIALDA® andROWASA®), corticosteroids/glucocorticoids (including prednisolonemethasulfobenzoate, tixocortol pivalate, fluticasone propionate,beclomethasone dipropionate, and budesonide), antibiotics such asmetronidazole or ciprofloxacin (or other antibiotics useful fortreating, for example, patients afflicted with fistulas), andimmunosuppressives such as azathioprine (for example, IMURAN® andAZASAN®), 6-mercaptopurine (for example, PURINETHOL®), methotrexate (forexample, TREXALL®, RHEUMATREX®), tacrolimus (for example, PROGRAF®) andcyclosporine (for example, GENGRAF®, NEORAL®, and SANDIMMUNE®). Suchagent(s) may be administered orally or by another route, for example viasuppository or enema, at dosages and intervals that are known in the artand described in the prescribing information.

Furthermore, IL23 antibodies or antibody derivatives, or the aforesaidcombinations, can be used in conjunction with one or more molecules orother treatments, wherein the other molecule(s) and/or treatment(s) donot directly bind to or affect IL23, but which combination is effectivefor treating or preventing the condition being treated. For example, ananti-IL23 antibody can be used in combination with probiotic therapy, orother therapy used to restore or maintain normal gut flora, includinggut flora transplant.

In one embodiment, one or more of the molecule(s) and/or treatment(s)treats or prevents a condition that is caused by one or more of theother molecule(s) or treatment(s) in the course of therapy, e.g.,nausea, fatigue, alopecia, cachexia, insomnia, etc. Such agent(s) ortherapies may be administered by routes, and at dosages and intervals,that are known in the art and described in the prescribing information.

In some aspects, IL23 antibodies or antibody derivatives, or theaforesaid combinations, can be used in conjunction with one or moreelemental diet treatment. See, e.g., Voitk et al. Arch Surg 107:329(1973); Yamamoto et al. Int J Colorectal Dis 28:335-340 (2013).

Additional supportive therapies are included in possible combinationtreatment with IL23 antagonists (e.g., an anti-IL23 antibody targeting,e.g., the p19 subunit of IL23, such as MEDI2070, or an antigen-bindingfragment thereof); such supportive therapies as (without limitation),analgesics, and anticholinergic and antidiarrheal agents. Combining suchsupportive therapies can be useful in the beginning of a treatmentregimen in reducing a patient's symptoms and improving their quality oflife. Supportive therapies include administering oral iron, folate, andvitamin B₁₂. Antidiarrheal agents include, but are not limited todiphenoxylate, codeine, loperamide, and anticholinergics (orpharmacological equivalents thereof), which can be administered topatients with mild disease to reduce the frequency of bowel movementsand relive rectal urgency. Cholestyramine can be used in patients toprevent bile salt-induced colonic secretion in patients who have alreadyundergone limited ileocolic resections. Anticholinergic agents include,but are not limited to, clidinium bromide, dicyclomine hydrochloride,tincture of belladonna and the like, and are useful to reduce abdominalcramps, pain and rectal urgency. Supportive or therapies may beadministered by routes, and at dosages and intervals, that are known inthe art and described in the prescribing information

In every case where a combination of molecules and/or other treatmentsis used, the individual molecule(s) and/or treatment(s) can beadministered in any order, over any length of time, which is effective,e.g., simultaneously, consecutively, or alternately. In one embodiment,the method of treatment comprises completing a first course of treatmentwith one molecule or other treatment before beginning a second course oftreatment. The length of time between the end of the first course oftreatment and beginning of the second course of treatment can be anylength of time that allows the total course of therapy to be effective,e.g., seconds, minutes, hours, days, weeks, months, or even years.

VII. Kits and Assays for Detecting IL23 Pathway Biomarkers

This disclosure also provides kits for detecting CCL20 alone or incombination with at least one IL23 pathway biomarker, e.g., IL22 and/orLCN2; at least one inflammation biomarker, e.g., CRP and/or FCP; or acombination thereof (e.g., protein expression level or gene expressionlevel), for example, through an immunoassay method. Such kits cancomprise containers, each with one or more of the various reagents(e.g., in concentrated form) utilized in the method, including, forexample, one or more anti-CCL20, anti-IL22, anti-LCN2, anti-CRP, and/oranti-FCP antibodies. One or more anti-CCL20, anti-IL22, anti-LCN2,anti-CRP, and/or anti-FCP antibodies, e.g., capture antibodies, can beprovided already attached to a solid support. One or more antibodiesanti-CCL20, anti-IL22, anti-LCN2, anti-CRP, and/or anti-FCP antibodies,e.g., detection antibodies, can be provided already conjugated to adetectable label, e.g., biotin or a ruthenium chelate.

The kit can also provide reagents for coupling a detectable label to anantibody (as well as the label itself), buffers, and/or reagents andinstrumentation to support the practice of the assays provided herein.In certain aspects, a labeled secondary antibody can be provided thatbinds to the detection antibody. A kit provided according to thisdisclosure can further comprise suitable containers, plates, and anyother reagents or materials necessary to practice the assays providedherein.

In some aspects, a kit comprises one or more nucleic acid probes (e.g.,oligonucleotides comprising naturally occurring and/or chemicallymodified nucleotide units) capable of hybridizing a subsequence of theCCL20, IL22, and/or LCN2 gene sequences under high stringencyconditions. In some aspects, one or more nucleic acid probes (e.g.,oligonucleotides comprising naturally occurring and/or chemicallymodified nucleotide units) capable of hybridizing a subsequence of theCCL20, IL22, and/or LCN2 gene sequence under high stringency conditionsare attached to a microarray chip.

A kit provided according to this disclosure can also comprise brochuresor instructions describing the process. For CCL20, IL22, and/or LCN2detection immunoassays, and in particular sandwich immunoassays, e.g.,an ELISA assay or an ECL assay, the sandwich immunoassay processcomprises a first anti-CCL20, anti-IL22, and/or anti-LCN2 “capture”antibody or antigen-binding fragment thereof attached to a solidsupport, and a second anti-CCL20, anti-IL22, and/or anti-LCN2“detection” antibody or antigen binding fragment thereof. Theimmunoassay can be performed by methods provided herein or methods wellknown and understood by those of ordinary skill in the art. In oneaspect, the immunoassay comprises attaching a capture antibody orfragment thereof to a solid support; applying the test sample or acontrol sample, allowing CCL20, IL22, and/or LCN2, if present in thesample, to bind to the capture antibody or fragment thereof; applyingthe detection antibody or fragment thereof, which can bind to CCL20,IL22, and/or LCN2 already bound to the capture antibody or fragmentthereof; and measuring the amount of detection antibody or fragmentthereof bound to CCL20, IL22, and/or LCN2. In certain aspects, the assaycan further include washing steps, blocking steps and incubation steps.

Test kits can include instructions for carrying out one or more CCL20,IL22, and/or LCN2 detection assays, e.g., immunoassays or nucleic aciddetection assays. Instructions included in the kits can be affixed topackaging material or can be included as a package insert. While theinstructions are typically written or printed materials they are notlimited to such. Any medium capable of storing such instructions andcommunicating them to an end user is contemplated. Such media include,but are not limited to, electronic storage media (e.g., magnetic discs,tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.As used herein, the term “instructions” can include the address of aninternet site that provides the instructions.

VIII. Companion Diagnostic System

The methods disclosed herein can be provided as a companion diagnostic,for example available via a web server, to inform the clinician orpatient about potential treatment choices. The methods disclosed hereincan comprise collecting or otherwise obtaining a biological sample andperforming an analytical method to detect and measure the levels ofCCL20 alone or in combination with at least one IL23 pathway biomarker,e.g., IL22 and/or LCN2; at least one inflammation biomarker, e.g., CRPand/or FCP; or a combination thereof (e.g., protein expression levels orgene expression levels of CCL20, IL22, LCN2, FCP, CRP, and/orcombinations thereof). Exemplary biomarkers that can be combined withCCL20 and combinations thereof are discussed above in the specification.

Levels of CCL20 alone, or in combination with the levels of one, two,three, or more biomarkers, including, e.g., IL22, LCN2, CRP, FCP, andcombinations thereof (e.g., protein expression levels or gene expressionlevels) or normalized scores derived from measured levels can be usedalone (e.g., for treatment, diagnostic, prognostic, or monitoringpurposes), or in combination with levels or normalized scores derivedfrom other biomarkers (e.g., a panel a genes used to derive a genesignature).

These scores can also be combined with other scores corresponding, forexample, to clinical biomarkers such as (i) gender, (ii) age, (iii) bodymass index, (iv) smoking status, (v) concomitant drugs, (vi) healthassessment quality (HAQ), or a combination of two or more to yield adiagnostic score. In this approach, the diagnostic score may be a singlenumber determined from the sum of all the marker calculations that iscompared to a preset threshold value that is an indication of thepresence or absence of disease. Or the diagnostic score may be a seriesof bars that each represent a biomarker value and the pattern of theresponses may be compared to a pre-set pattern for determination of thepresence or absence of disease.

At least some aspects of the methods described herein, due to thecomplexity of the calculations involved, a method comprising the use ofCCL20 alone or in combination with other biomarkers, including, e.g.,IL22, LCN2, CRP, FCP, and combinations thereof can be implemented withthe use of a computer. In some aspects, the computer system compriseshardware elements that are electrically coupled via bus, including aprocessor, input device, output device, storage device,computer-readable storage media reader, communications system,processing acceleration (e.g., DSP or special-purpose processors), andmemory.

The computer-readable storage media reader can be further coupled tocomputer-readable storage media, the combination comprehensivelyrepresenting remote, local, fixed and/or removable storage devices plusstorage media, memory, etc. for temporarily and/or more permanentlycontaining computer-readable information, which can include storagedevice, memory and/or any other such accessible system resource.

A single architecture might be utilized to implement one or more serversthat can be further configured in accordance with currently desirableprotocols, protocol variations, extensions, etc. However, it will beapparent to those skilled in the art that embodiments may well beutilized in accordance with more specific application requirements.Customized hardware might also be utilized and/or particular elementsmight be implemented in hardware, software or both. Further, whileconnection to other computing devices such as network input/outputdevices (not shown) may be employed, it is to be understood that wired,wireless, modem, and/or other connection or connections to othercomputing devices might also be utilized.

In one aspect, the system further comprises one or more devices forproviding input data to the one or more processors. The system furthercomprises a memory for storing a data set of ranked data elements. Inanother aspect, the device for providing input data comprises a detectorfor detecting the characteristic of the data element, e.g., such as afluorescent plate reader, mass spectrometer, or gene chip reader.

The system additionally may comprise a database management system. Userrequests or queries can be formatted in an appropriate languageunderstood by the database management system that processes the query toextract the relevant information from the database of training sets. Thesystem may be connectable to a network to which a network server and oneor more clients are connected. The network may be a local area network(LAN) or a wide area network (WAN), as is known in the art. Preferably,the server includes the hardware necessary for running computer programproducts (e.g., software) to access database data for processing userrequests. The system can be in communication with an input device forproviding data regarding data elements to the system (e.g., expressionvalues). In one aspect, the input device can include a gene expressionprofiling system including, e.g., a mass spectrometer, gene chip orarray reader, and the like.

Some aspects described herein can be implemented so as to include acomputer program product. A computer program product may include acomputer readable medium having computer readable program code embodiedin the medium for causing an application program to execute on acomputer with a database. As used herein, a “computer program product”refers to an organized set of instructions in the form of natural orprogramming language statements that are contained on a physical mediaof any nature (e.g., written, electronic, magnetic, optical orotherwise) and that may be used with a computer or other automated dataprocessing system. Such programming language statements, when executedby a computer or data processing system, cause the computer or dataprocessing system to act in accordance with the particular content ofthe statements.

Computer program products include without limitation: programs in sourceand object code and/or test or data libraries embedded in a computerreadable medium. Furthermore, the computer program product that enablesa computer system or data processing equipment device to act inpre-selected ways may be provided in a number of forms, including, butnot limited to, original source code, assembly code, object code,machine language, encrypted or compressed versions of the foregoing andany and all equivalents.

In one aspect, a computer program product is provided to implemented thetreatment, diagnostic, prognostic, or monitoring methods disclosedherein, for example, to determine whether to administer an IL23antagonist (e.g., an anti-IL23 antibody targeting, e.g., the p19 subunitof IL23, such as MEDI2070, or an antigen-binding fragment thereof) to apatient in need thereof if the level of CCL20 in a sample taken from thepatient is above or below a predetermined threshold level.

In one aspect, a computer program product is provided to implement thetreatment, diagnostic, prognostic, or monitoring methods disclosedherein, for example, to determine whether to administer an IL23antagonist (e.g., an anti-IL23 antibody targeting, e.g., the p19 subunitof IL23, such as MEDI2070, or an antigen-binding fragment thereof) to apatient in need thereof if the levels of CCL20 and the levels of one,two, three, or more additional biomarkers, e.g., IL22, LCN2, CRP, and/orFCP, in a sample taken from the patient are above or below theirrespective predetermined threshold level.

In some aspects, the IL23 antagonist is an anti-IL23 antibody which canspecifically bind to the p19 subunit of IL23 (SEQ ID NO: 15), to the p40subunit of IL23 (SEQ ID NO: 16), or both.

In some aspects, the anti-IL23 antibody or antigen-binding fragmentthereof comprises the heavy chain (HC) (SEQ ID NO: 17) and/or the lightchain (SEQ ID NO: 18) of MEDI2070, or an antigen-binding fragment,variant, or derivative thereof. In some aspects, the anti-IL23 antibodyor antigen-binding fragment thereof comprises the heavy chain variableregion (VH) (SEQ ID NO: 7) and/or the light chain variable region (VL)(SEQ ID NO: 8) of MEDI2070. In some aspects, the anti-IL23 antibody orantigen-binding fragment thereof comprises at least one of thecomplementarity determining regions of MEDI2070 (SEQ ID NOS: 33-38). Insome aspects, the anti-IL23 antibody or antigen-binding fragment thereofcomprises a VH region comprising the sequence of SEQ ID NO: 45 and/or aVL region comprising the sequence of SEQ ID NO: 46, or anantigen-binding fragment, variant, or derivative thereof. In someaspects, the anti-IL23 antibody or antigen-binding fragment thereofcomprises at least one of the complementarity determining regions of SEQID NOS: 47-49 (CDRs of the VH of SEQ ID NO: 45) and/or SEQ ID NOS: 50-52(CDRs of the VL of SEQ ID NO: 46).

In other aspects, the IL23 antagonist is an anti-IL23 antibody selectedfrom ustekinumab or briakinumab (targeting the p40 subunit of IL23),guselkumab, tildrakizumab, BI-655066 or LY-3074828 (targeting the p19subunit of IL23), an antigen-binding fragment thereof, or a combinationthereof. In other aspects, the IL23 antagonist is a molecule (e.g., anantibody) that competes for binding to IL23 with ustekinumab orbriakinumab (targeting the p40 subunit of IL23), guselkumab,tildrakizumab, BI-655066 or LY-3074828 (targeting the p19 subunit ofIL23), an antigen-binding fragment thereof, or a combination thereof.

The computer program product includes a computer readable mediumembodying program code executable by a processor of a computing deviceor system, the program code comprising: (a) code that retrieves dataattributed to a biological sample from a subject, wherein the datacomprises CCL20 levels values (presence/absence, amount, or dataotherwise derived from these level values) alone or combination withvalues corresponding to other biomarkers in the biological sample (e.g.,biological markers or clinical markers) such as IL22, LCN2, FCP, CRP, orcombinations thereof. These values can also be combined with valuescorresponding to clinical biomarkers, for example, indicative of theseverity of the disease or disorder or to the patient's predispositionto have such disease or disorder; and, (b) code that executes aclassification method that indicates, e.g., whether to administer anIL23 antagonist (e.g., an anti-IL23 antibody targeting, e.g., the p19subunit of IL23, such as MEDI2070, or an antigen-binding fragmentthereof) to a patient in need thereof.

While various aspects have been described as methods or apparatuses, itshould be understood that aspects can be implemented through codecoupled with a computer, e.g., code resident on a computer or accessibleby the computer. For example, software and databases could be utilizedto implement many of the methods discussed above. Thus, in addition toaspects accomplished by hardware, it is also noted that these aspectscan be accomplished through the use of an article of manufacturecomprised of a computer usable medium having a computer readable programcode embodied therein, which causes the enablement of the functionsdisclosed in this description. Therefore, it is desired that aspectsalso be considered protected by this patent in their program code meansas well.

Furthermore, some aspects can be code stored in a computer-readablememory of virtually any kind including, without limitation, RAM, ROM,magnetic media, optical media, or magneto-optical media. Even moregenerally, some aspects could be implemented in software, or inhardware, or any combination thereof including, but not limited to,software running on a general purpose processor, microcode, PLAs, orASICs.

It is also envisioned that some aspects could be accomplished ascomputer signals embodied in a carrier wave, as well as signals (e.g.,electrical and optical) propagated through a transmission medium. Thus,the various types of information discussed above could be formatted in astructure, such as a data structure, and transmitted as an electricalsignal through a transmission medium or stored on a computer readablemedium.

List of Abbreviations

Abbreviation or Specialized Term Definition ADA(s) anti-drugantibody/antibodies AE adverse event ANCOVA analysis of covariance ASankylosing spondylitis CD Crohn's disease CDAI Crohn's Disease ActivityIndex CRP C-reactive protein DNA deoxyribonucleic acid GIGastrointestinal IBD inflammatory bowel disease IBDQ inflammatory boweldisease questionnaire IFN Interferon IgG2 immunoglobulin G2 ILInterleukin IM Immunogenicity IV Intravenous LOCF last observationcarried forward Mab monoclonal antibody MedDRA Medical Dictionary forRegulatory Activities mITT modified intent-to-treat MS multiplesclerosis NK natural killer PD Pharmacodynamics PK pharmacokinetic(s) PPper protocol PROs Patient Reported Outcomes PsO Psoriasis Q4W every 4weeks RA rheumatoid arthritis RNA ribonucleic acid SAE serious adverseevent SC subcutaneous(ly) Th T helper TNFα tumor necrosis factor-alphaUC ulcerative colitis w/v weight per volume

Sequences

SEQ ID NO Description Sequence 1 CCL20MCCTKSLLLAALMSVLLLHLCGESEAASNFDCCLGYTDRILHPKFIVGFT proteinRQLANEGCDINAIIFHTKKKLSVCANPKQTWVKYIVRLLSKKVKNM 2 CCL20AGAATATAACAGCACTCCCAAAGAACTGGGTACTCAACACTGAGCAGATC geneTGTTCTTTGAGCTAAAAACCATGTGCTGTACCAAGAGTTTGCTCCTGGCT (mRNA)GCTTTGATGTCAGTGCTGCTACTCCACCTCTGCGGCGAATCAGAAGCAGCAAGCAACTTTGACTGCTGTCTTGGATACACAGACCGTATTCTTCATCCTAAATTTATTGTGGGCTTCACACGGCAGCTGGCCAATGAAGGCTGTGACATCAATGCTATCATCTTTCACACAAAGAAAAAGTTGTCTGTGTGCGCAAATCCAAAACAGACTTGGGTGAAATATATTGTGCGTCTCCTCAGTAAAAAAGTCAAGAACATGTAAAAACTGTGGCTTTTCTGGAATGGAATTGGACATAGCCCAAGAACAGAAAGAACCTTGCTGGGGTTGGAGGTTTCACTTGCACATCATGGAGGGTTTAGTGCTTATCTAATTTGTGCCTCACTGGACTTGTCCAATTAATGAAGTTGATTCATATTGCATCATAGTTTGCTTTGTTTAAGCATCACATTAAAGTTAAACTGTATTTTATGTTATTTATAGCTGTAGGTTTTCTGTGTTTAGCTATTTAATACTAATTTTCCATAAGCTATTTTGGTTTAGTGCAAAGTATAAAATTATATTTGGGGGGGAATAAGATTATATGGACTTTCTTGCAAGCAACAAGCTATTTTTTAAAAAAAACTATTTAACATTCTTTTGTTTATATTGTTTTGTCTCCTAAATTGTTGTAATTGCATTATAAAATAAGAAAAATATTAATAAGACAAATATTGAAAATAAAGAAACAAAAAGTTCTTCTGTTAAAAAAAA A 3 IL22 geneCGACCAGGTTCTCCTTCCCCAGTCACCAGTTGCTCGAGTTAGAATTGTCT (mRNA)GCAATGGCCGCCCTGCAGAAATCTGTGAGCTCTTTCCTTATGGGGACCCTGGCCACCAGCTGCCTCCTTCTCTTGGCCCTCTTGGTACAGGGAGGAGCAGCTGCGCCCATCAGCTCCCACTGCAGGCTTGACAAGTCCAACTTCCAGCAGCCCTATATCACCAACCGCACCTTCATGCTGGCTAAGGAGGCTAGCTTGGCTGATAACAACACAGACGTTCGTCTCATTGGGGAGAAACTGTTCCACGGAGTCAGTATGAGTGAGCGCTGCTATCTGATGAAGCAGGTGCTGAACTTCACCCTTGAAGAAGTGCTGTTCCCTCAATCTGATAGGTTCCAGCCTTATATGCAGGAGGTGGTGCCCTTCCTGGCCAGGCTCAGCAACAGGCTAAGCACATGTCATATTGAAGGTGATGACCTGCATATCCAGAGGAATGTGCAAAAGCTGAAGGACACAGTGAAAAAGCTTGGAGAGAGTGGAGAGATCAAAGCAATTGGAGAACTGGATTTGCTGTTTATGTCTCTGAGAAATGCCTGCATTTGACCAGAGCAAAGCTGAAAAATGAATAACTAACCCCCTTTCCCTGCTAGAAATAACAATTAGATGCCCCAAAGCGATTTTTTTTAACCAAAAGGAAGATGGGAAGCCAAACTCCATCATGATGGGTGGATTCCAAATGAACCCCTGCGTTAGTTACAAAGGAAACCAATGCCACTTTTGTTTATAAGACCAGAAGGTAGACTTTCTAAGCATAGATATTTATTGATAACATTTCATTGTAACTGGTGTTCTATACACAGAAAACAATTTATTTTTTAAATAATTGTCTTTTTCCATAAAAAAGATTACTTTCCATTCCTTTAGGGGAAAAAACCCCTAAATAGCTTCATGTTTCCATAATCAGTACTTTATATTTATAAATGTATTTATTATTATTATAAGACTGCATTTTATTTATATCATTTTATTAATATGGATTTATTTATAGAAACATCATTCGATATTGCTACTTGAGTGTAAGGCTAATATTGATATTTATGACAATAATTATAGAGCTATAACATGTTTATTTGACCTCAATAAACACTTGGATATCC 4 IL22 proteinMAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDREQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI 5 LCN2 geneACTCGCCACCTCCTCTTCCACCCCTGCCAGGCCCAGCAGCCACCACAGCG (mRNA)CCTGCTTCCTCGGCCCTGAAATCATGCCCCTAGGTCTCCTGTGGCTGGGCCTAGCCCTGTTGGGGGCTCTGCATGCCCAGGCCCAGGACTCCACCTCAGACCTGATCCCAGCCCCACCTCTGAGCAAGGTCCCTCTGCAGCAGAACTTCCAGGACAACCAATTCCAGGGGAAGTGGTATGTGGTAGGCCTGGCAGGGAATGCAATTCTCAGAGAAGACAAAGACCCGCAAAAGATGTATGCCACCATCTATGAGCTGAAAGAAGACAAGAGCTACAATGTCACCTCCGTCCTGTTTAGGAAAAAGAAGTGTGACTACTGGATCAGGACTTTTGTTCCAGGTTGCCAGCCCGGCGAGTTCACGCTGGGCAACATTAAGAGTTACCCTGGATTAACGAGTTACCTCGTCCGAGTGGTGAGCACCAACTACAACCAGCATGCTATGGTGTTCTTCAAGAAAGTTTCTCAAAACAGGGAGTACTTCAAGATCACCCTCTACGGGAGAACCAAGGAGCTGACTTCGGAACTAAAGGAGAACTTCATCCGCTTCTCCAAATCTCTGGGCCTCCCTGAAAACCACATCGTCTTCCCTGTCCCAATCGACCAGTGTATCGACGGCTGAGTGCACAGGTGCCGCCAGCTGCCGCACCAGCCCGAACACCATTGAGGGAGCTGGGAGACCCTCCCCACAGTGCCACCCATGCAGCTGCTCCCCAGGCCACCCCGCTGATGGAGCCCCACCTTGTCTGCTAAATAAACATGTGCCCTCAGGCCAAAAAAAAAAAAAAAAAA 6 LCN2MPLGLLWLGLALLGALHAQAQDSTSDLIPAPPLSKVPLQQNFQDNQFQGK proteinWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG 7 MEDI2070QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAV VHIWYDGSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWGQGTMVTVSS 8 MEDI2070QSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLI VLYGSGNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGW VFGGGTRLTVL 9CRP gene AAGGCAAGAGATCTAGGACTTCTAGCCCCTGAACTTTCAGCCGAATACAT (mRNA)CTTTTCCAAAGGAGTGAATTCAGGCCCTTGTATCACTGGCAGCAGGACGTGACCATGGAGAAGCTGTTGTGTTTCTTGGTCTTGACCAGCCTCTCTCATGCTTTTGGCCAGACAGACATGTCGAGGAAGGCTTTTGTGTTTCCCAAAGAGTCGGATACTTCCTATGTATCCCTCAAAGCACCGTTAACGAAGCCTCTCAAAGCCTTCACTGTGTGCCTCCACTTCTACACGGAACTGTCCTCGACCCGTGGGTACAGTATTTTCTCGTATGCCACCAAGAGACAAGACAATGAGATTCTCATATTTTGGTCTAAGGATATAGGATACAGTTTTACAGTGGGTGGGTCTGAAATATTATTCGAGGTTCCTGAAGTCACAGTAGCTCCAGTACACATTTGTACAAGCTGGGAGTCCGCCTCAGGGATCGTGGAGTTCTGGGTAGATGGGAAGCCCAGGGTGAGGAAGAGTCTGAAGAAGGGATACACTGTGGGGGCAGAAGCAAGCATCATCTTGGGGCAGGAGCAGGATTCCTTCGGTGGGAACTTTGAAGGAAGCCAGTCCCTGGTGGGAGACATTGGAAATGTGAACATGTGGGACTTTGTGCTGTCACCAGATGAGATTAACACCATCTATCTTGGCGGGCCCTTCAGTCCTAATGTCCTGAACTGGCGGGCACTGAAGTATGAAGTGCAAGGCGAAGTGTTCACCAAACCCCAGCTGTGGCCCTGAGGCCCAGCTGTGGGTCCTGAAGGTACCTCCCGGTTTTTTACACCGCATGGGCCCCACGTCTCTGTCTCTGGTACCTCCCGCTTTTTTACACTGCATGGTTCCCACGTCTCTGTCTCTGGGCCTTTGTTCCCCTATATGCATTGCAGGCCTGCTCCACCCTCCTCAGCGCCTGAGAATGGAGGTAAAGTGTCTGGTCTGGGAGCTCGTTAACTATGCTGGGAAACGGTCCAAAAGAATCAGAATTTGAGGTGTTTTGTTTTCATTTTTATTTCAAGTTGGACAGATCTTGGAGATAATTTCTTACCTCACATAGATGAGAAAACTAACACCCAGAAAGGAGAAATGATGTTATAAAAAACTCATAAGGCAAGAGCTGAGAAGGAAGCGCTGATCTTCTATTTAATTCCCCACCCATGACCCCCAGAAAGCAGGAGGGCATTGCCCACATTCACAGGGCTCTTCAGTCTCAGAATCAGGACACTGGCCAGGTGTCTGGTTTGGGTCCAGAGTGCTCATCATCATGTCATAGAACTGCTGGGCCCAGGTCTCCTGAAATGGGAAGCCCAGCAATACCACGCAGTCCCTCCACTTTCTCAAAGCACACTGGAAAGGCCATTAGAATTGCCCCAGCAGAGCAGATCTGCTTTTTTTCCAGAGCAAAATGAAGCACTAGGTATAAATATGTTGTTACTGCCAAGAACTTAAATGACTGGTTTTTGTTTGCTTGCAGTGCTTTCTTAATTTTATGGCTCTTCTGGGAAACTCCTCCCCTTTTCCACACGAACCTTGTGGGGCTGTGAATTCTTTCTTCATCCCCGCATTCCCAATATACCCAGGCCACAAGAGTGGACGTGAACCACAGGGTGTCCTGTCAGAGGAGCCCATCTCCCATCTCCCCAGCTCCCTATCTGGAGGATAGTTGGATAGTTACGTGTTCCTAGCAGGACCAACTACAGTCTTCCCAAGGATTGAGTTATGGACTTTGGGAGTGAGACATCTTCTTGCTGCTGGATTTCCAAGCTGAGAGGACGTGAACCTGGGACCACCAGTAGCCATCTTGTTTGCCACATGGAGAGAGACTGTGAGGACAGAAGCCAAACTGGAAGTGGAGGAGCCAAGGGATTGACAAACAACAGAGCCTTGACCACGTGGAGTCTCTGAATCAGCCTTGTCTGGAACCAGATCTACACCTGGACTGCCCAGGTCTATAAGCCAATAAAGCCCCTGTTTACTTGAAAAAAAAAA 10 CRP proteinMEKLLCFLVLTSLSHAFGQTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLHFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHICTSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP 11 CalprotectinATGTCTCTTGTCAGCTGTCTTTCAGAAGACCTGGTGGGGCAAGTCCGTGG S100A8GCATCATGTTGACCGAGCTGGAGAAAGCCTTGAACTCTATCATCGACGTC subunitTACCACAAGTACTCCCTGATAAAGGGGAATTTCCATGCCGTCTACAGGGA (mRNA)TGACCTGAAGAAATTGCTAGAGACCGAGTGTCCTCAGTATATCAGGAAAAAGGGTGCAGACGTCTGGTTCAAAGAGTTGGATATCAACACTGATGGTGCAGTTAACTTCCAGGAGTTCCTCATTCTGGTGATAAAGATGGGCGTGGCAGCCCACAAAAAAAGCCATGAAGAAAGCCACAAAGAGTAGCTGAGTTACTGGGCCCAGAGGCTGGGCCCCTGGACATGTACCTGCAGAATAATAAAGTCATCAATACCTCAAAAAAAAAAAAAAAAAAAAA 12 CalprotectinMLTELEKALNSIIDVYHKYSLIKGNFHAVYRDDLKKLLETECPQYIRKKG S100A8ADVWFKELDINTDGAVNFQEFLILVIKMGVAAHKKSHEE SHKE subunit 13 CalprotectinAAACACTCTGTGTGGCTCCTCGGCTTTGACAGAGTGCAAGACGATGACTT S100A9GCAAAATGTCGCAGCTGGAACGCAACATAGAGACCATCATCAACACCTTC subunitCACCAATACTCTGTGAAGCTGGGGCACCCAGACACCCTGAACCAGGGGGA (mRNA)ATTCAAAGAGCTGGTGCGAAAAGATCTGCAAAATTTTCTCAAGAAGGAGAATAAGAATGAAAAGGTCATAGAACACATCATGGAGGACCTGGACACAAATGCAGACAAGCAGCTGAGCTTCGAGGAGTTCATCATGCTGATGGCGAGGCTAACCTGGGCCTCCCACGAGAAGATGCACGAGGGTGACGAGGGCCCTGGCCACCACCATAAGCCAGGCCTCGGGGAGGGCACCCCCTAAGACCACAGTGGCCAAGATCACAGTGGCCACGGCCACGGCCACAGTCATGGTGGCCACGGCCACAGCCACTAATCAGGAGGCCAGGCCACCCTGCCTCTACCCAACCAGGGCCCCGGGGCCTGTTATGTCAAACTGTCTTGGCTGTGGGGCTAGGGGCTGGGGCCAAATAAAGTCTCTTCCTCCAAGTCAAAAAAAAAA 14 CalprotectinMTCKMSQLERNIETIINTFHQYSVKLGHPDTLNQGEFKELVRKDLQNFLK S100A9KENKNEKVIEHIMEDLDTNADKQLSFEEFIMLMARLTWASHEKMHEGDEG subunitPGHHHKPGLGEGTP 15 IL-23 alphaMLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEGsubunit (p19)DEETTNDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGEPSLLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQAFVAVAARVF AHGAATLSP16 IL-12 betaMCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWsubunit (p40)TLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS 17 MEDI2070QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNEYYAD HCSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYTSSWYPDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 18 MEDI2070QSVLTQPPSVSGAPGQRVTISCTGSSSNTGAGYDVHWYQQVPGTAPKLLIYGSGNRPSGVPD LCRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTRLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 19 Ustekinumab EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWLGWVRQMPGKGLDWIGIMSPVDSDIRY HCSPSFQGQVTMSVDKSITTAYLQWNSLKASDTAMYYCARRRPGQGYFDFWGQGTLVTVSSSSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 20 UstekinumabDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPS LCRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 21 VedolizumabQVQLVQSGAEVKKPGASVKVSCKGSGYTFTSYWMHWVRQAPGQRLEWIGEIDPSESNTNY HCNQKFKGRVTLTVDISASTAYMELSSLRSEDTAVYYCARGGYDGWDYAIDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 22 Vedolizumab DVVMTQSPLSLPVTPGEPASISCRSSQSLAKSYGNTYLSWYLQKPGQSPQLLIYGISNRF LCSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGTHQPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 23 AdalimumabEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDY HCADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 24 AdalimumabDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPS LCRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 25 CertolizumabEVQLVESGGGLVQPGGSLRLSCAASGYVFTDYGMNWVRQAPGKGLEWMGWINTYIGEPIY HCADSVKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARGYRSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCAA 26 CertolizumabDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSASFLYSGVPY LCRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 27 BriakinumabQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYY HCADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 28 BriakinumabQSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVP LCDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDRYTHPALLEGTGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 29 TildrakizumabQVQLVQSGAEVKKPGASVKVSCKASGYIFITYWMTWVRQAPGQGLEWMGQIFPASGSADY HCNEKFEGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGGGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 30 TildrakizumabDIQMTQSPSSLSASVGDRVTITCRTSENIYSYLAWYQQKPGKAPKLLIYNAKTLAEGVPS LCRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGIPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 31 EtrolizumabEVQLVESGGGLVQPGGSLRLSCAASGFFITNNYWGWVRQAPGKGLEWVGYISYSGSTSYN HCPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARTGSSGYFDFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 32 EtrolizumabDIQMTQSPSSLSASVGDRVTITCRASESVDDLLHWYQQKPGKAPKLLIKYASQSISGVPS LCRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNSLPNTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 33 MEDI2070 SYGMH VH-CDR1 34 MEDI2070VIWYDGSNEYYADSVKG VH-CDR2 35 MEDI207- DPGYLSSWYPDAFDI VH-CDR3 36MEDI2070 IGSSSNIGAGYDVH VL-CDR1 37 MEDI2070 GSGNRPS VL-CDR2 38 MEDI2070QSYDSSLSGWV VL-CDR3 39 LCN2 DQCIDG variant 40 LCN2 GNGQSG variant 41CRP variant YSIFSYATKRQDNEIL 42 CRP variant TVFSRMPPRDKTMRFF 43 S100A8VAAHKKSHEESHKE variant 44 S100A8 WQPTKKAMKKATKSS variant 45 Antibody BQVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGHIHYSGNTYYN VHPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAKNRGFYYGMDVWGQGTTVTVSS 46 Antibody BDIQMTQSPSSVSASVGDRVTITCRASQVISSWLAWYQQKPGKAPSLLIYAASSLQSGVPSRF VLSGSVSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVDFK 47 Antibody B SGGYYWSVH-CDR1 48 Antibody B HIHYSGNTYYNPSLKS VH-CDR2 49 Antibody B NRGFYYGMDVVH-CDR3 50 Antibody B RASQVISSWLA VL-CDR1 51 Antibody B AASSLQS VL-CDR252 Antibody B QQANSFPFT VL-CDR3

All patents and publications referred to herein are expresslyincorporated by reference in their entireties.

Aspects of the present disclosure can be further defined by reference tothe following non-limiting examples, which describe in detailpreparation of certain antibodies of the present disclosure and methodsfor using antibodies of the present disclosure. It will be apparent tothose skilled in the art that many modifications, both to materials andmethods, can be practiced without departing from the scope of thepresent disclosure.

It is to be appreciated that the Detailed Description section, and notthe Summary and Abstract sections, is intended to be used to interpretthe claims. The Summary and Abstract sections may set forth one or morebut not all exemplary aspects of the present invention as contemplatedby the inventor(s), and thus, are not intended to limit the presentinvention and the appended claims in any way.

The present invention has been described above with the aid offunctional building blocks illustrating the implementation of specifiedfunctions and relationships thereof. The boundaries of these functionalbuilding blocks have been arbitrarily defined herein for the convenienceof the description. Alternate boundaries can be defined so long as thespecified functions and relationships thereof are appropriatelyperformed.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples which detail exemplary aspects of the invention.

Example 1 Phase 2a Clinical Study Protocol

A Phase 2a study was conducted to evaluate the efficacy and safety ofmultiple IV doses of MEDI2070 (700 mg) or placebo administered on Week 0(Day 1) and Week 4 (Day 29) during an initial 12-week treatment periodin subjects with moderate to severe CD who have failed or are intolerantto anti-TNFα therapy. The following clinical trial protocol was carriedout in human patients. Results of the clinical trial are presented inExample 2.

I. Clinical Study Summary

The Phase 2a study evaluated the efficacy and safety of multiple IVdoses of MEDI2070 (700 mg) or placebo administered on Week 0 (Day 1) andWeek 4 (Day 29) during an initial 12-week treatment period in subjectswith moderate to severe CD who failed or are intolerant to anti-TNFαtherapy. A 100-week, open-label, treatment period was included to allowevaluation of the long-term safety of MEDI2070 (administered at 210 mgSC Q4W) and to provide information on PK and efficacy data.

The primary objective of this study was to evaluate the efficacy ofMEDI2070 versus placebo to induce a clinical effect (defined as at leasta 100-point reduction in CDAI from baseline) or remission (defined asCDAI<150)) at Week 8 in subjects with moderate to severe CD.

Secondary objectives of this study included evaluating: the efficacy ofMEDI2070 versus placebo in achieving CDAI remission at Week 8; theeffect of MEDI2070 versus placebo in achieving at least a CDAI 100-pointreduction from baseline at Week 8; the effect of MEDI2070 versus placeboin achieving at least a CDAI 70-point reduction from baseline at Week 8;the effect of MEDI2070 versus placebo in achieving CDAI remission or atleast a CDAI 100-point reduction from baseline at Week 12; the effect ofMEDI2070 versus placebo on the change from baseline in CDAI at Week 8;the safety and tolerability of MEDI2070; and the PK and immunogenicity(IM) of MEDI2070.

Exploratory objectives of this study included evaluating: the effect ofMEDI2070 on other measures of clinical effect including but not limitedto CDAI response, change from baseline in CDAI at other timepoints, andsustained CDAI response; the effect of treatment on inflammatory markersin blood and stool; the predictive value of blood or fecal biomarkerswith respect to subject response to MEDI2070; and the effect of MEDI2070on Patient Reported Outcomes (PROs).

II. Study Design Overview

This was a two-part Phase 2a study comprising a 12-week, double-blind,placebo-controlled, treatment period followed by a 100-week, open-label,treatment period to evaluate the short-term efficacy, and the short- andlong-term safety of MEDI2070 in subjects with moderate to severe, activeCD who failed or are intolerant to anti-TNFα therapy as determined bythe investigator. Subjects were stratified based on the number of prioranti-TNFα agents that they have failed (1 vs >1). Subjects at variouscenters worldwide were randomized in a 1:1 ratio to initially receive afixed IV dose of MEDI2070 (700 mg) or placebo on Week 0 (Day 1) and Week4 (Day 29) during the 12-week, double-blind, placebo-controlled,treatment period. At the completion of the double-blind,placebo-controlled, treatment period (Week 12), subjects had the optionto enter a 100-week, open-label, treatment period where they receivedopen-label MEDI2070 (210 mg SC) Q4W (Week 12 through Week 112) asdescribed in FIG. 1.

MEDI2070 or placebo was administered as an IV infusion over a period ofat least 60 minutes via an infusion pump on Week 0 (Day 1) and Week 4(Day 29). Subjects who completed the 12-week, double-blind,placebo-controlled, treatment period and entered the 100-week,open-label, treatment period received MEDI2070 at a fixed dose of 210 mgSC injection Q4W for 26 doses (through Week 112). The primary analysisof the study was conducted after the last subject in the study completedthe 12-week, double-blind, placebo-controlled, treatment period or waswithdrawn from the study prior to completing the 12-week, double-blind,placebo-controlled, treatment period.

The doses of MEDI2070 in this study were 700 mg IV Q4W in the 12-week,double-blind, placebo-controlled, treatment period (Weeks 0 and 4) and210 mg SC Q4W in the 100-week, open-label, treatment period (Week 12 toWeek 112).

Pharmacokinetic/PD modeling of MEDI2070 predicted a greater than 99%mean suppression of plasma IL23 throughout the duration of the study forboth the 700 mg IV and 210 mg SC dosing regimens. Furthermore,(potency-corrected) serum concentrations of MEDI2070 at these dosingregimens were predicted to be higher than those of the anti-IL12/23p40antibody ustekinumab shown to be efficacious in CD (Sandborn et al.Gastroenterol. 135:1130-41 (2008)). Administration of 700 mg IV waspredicted to rapidly achieve steady-state target suppression.

Previous clinical data (Sandborn et al. Gastroenterol. 135:1130-41(2008)) indicate that changes from placebo in the response parametersafter administration of IV ustekinumab maximized between 6-8 weeks afterstart of ustekinumab administration; therefore, the proposedplacebo-controlled, double-blind period of 12 weeks is sufficient tocharacterize the effect of MEDI2070 in this patient population.Regulatory guidance recommends assessing the primary endpoint forinduction of effect between 4-8 weeks (or after 2 cycles of dosing),thus the assessment of the primary endpoint at Week 8 is appropriate. Inaddition, Week 12 was assessed to observe the potential extended timecourse of effect for MEDI2070.

All subjects received investigational product (700 mg MEDI2070 orplacebo) by IV infusion over a minimum period of 60 minutes during the12-week, double-blind, placebo-controlled, treatment period. MEDI2070(700 mg) or placebo was delivered in 5.0% w/v dextrose in a volume of100 mL over a minimum of 60 minutes using an infusion pump. Before andafter each IV infusion, the IV access was flushed with 5% w/v dextrose.

MEDI2070 was administered by subcutaneous (SC) injection during the100-week, open-label, treatment period. Each SC dose was administered tothe subject's anterior abdominal wall by an experienced and qualifiedstaff member. Each SC injection was no more than 1.0 mL in volume (i.e.,3×1.0 mL injections for the 210 mg SC dose). As the MEDI2070 dosagevolume exceeds 1.0 mL, the dose was equally divided in 3 syringes andadministered as multiple SC injections on alternating (left or right)sites on the subject's anterior abdominal wall with no more thanapproximately 30 seconds of time between each injection and at adistance of at least 2 cm apart.

III. Disease Evaluation and Methods

CDAI: The CDAI is the oldest and most widely used of several multi-iteminstruments that have been developed and is validated for use inclinical studies to measure disease activity in CD (Best et al.Gastroenterology 70:439-44 (1976); Sands et al. N Engl J Med.350(9):876-85 (2004)). The CDAI measures the severity of active diseaseusing symptom scores that are monitored over the previous week andincludes patient-reported symptoms, physician-assessed signs, andlaboratory markers. The CDAI score is calculated by summing weightedscores for subjective items (number of liquid or very soft stools,abdominal pain and general well-being) recorded by a diary during a1-week period, and objective items (associated symptoms, takingantidiarrheal such as loperamide/opiates, abdominal mass, hematocrit,daily morning temperature, and body weight). The CDAI scores range from0 to 600, with higher scores indicating greater disease activity.Subjects with scores of <150, 150 to 219, and 220 to 450 representremission, mild disease, and moderate to severe disease, respectively;whereas subjects with scores of >450 have very severe disease (Buxton etal. Value Health. 10:214-20 (2007)). The CDAI was calculated at the sitein order to determine the eligibility for the study. For statisticalanalysis, CDAI for all visits was also calculated.

Patient Reported Outcomes: The study assessed patient reported outcomes(PROs) using the IBDQ. The IBDQ was completed by subjects using a paperquestionnaire at study visits, scheduled at Weeks 0, 4, 8, and 12 andwas administered before any other assessments.

Inflammatory Bowel Disease Questionnaire (IBDQ): The IBDQ is avalidated, disease-specific PRO instrument that measures health-relatedquality of life in patients with IBD (Guyatt et al. Gastroenterology.96:804-10 (1989)). The IBDQ covers the following dimensions: bowelsymptoms (10 items), systemic symptoms (5 items), emotional function (12items), and social function (5 items). Items are scored on a 7-pointLikert scale, yielding a global score in the range 32 to 224 (withhigher scores indicating better quality of life). The IBDQ has beenfrequently used in drug approval applications to assess treatmentefficacy in IBD. The IBDQ was designed to be self-administered andcompleted in 5 minutes.

IV. Endpoints

The primary endpoint of the study was CDAI response at Week 8, definedby either a CDAI score of <150 or a CDAI reduction from baseline of atleast 100 points. Baseline was defined as the latest nonmissingobservation prior to first administration of the investigationalproduct.

Missing data was imputed by nonresponder imputation approach; i.e., anysubject with missing information on primary endpoint was assumed asnonresponder. In addition, subjects with a clinically meaningfulincrease in steroid use were assumed to be a nonresponder for theprimary analysis perspective.

For the primary endpoint, comparisons between treatment arms were basedon the mITT population and were performed using a logistic regressionmodel with treatment and stratification factor as covariate. Thestratification factor for this study was defined by the number of prioranti-TNFα agents a subject had received (1 vs >1). The significance oftreatment effect was tested using the two-sided test at a=10%.

The following sensitivity analyses were performed: (1) A sensitivityanalysis was performed by adjusting for baseline CDAI score and/or otherbaseline covariates by extending the model planned above for subjects inmITT population. (2) A mixed effect longitudinal logistic regressionmodel was implemented on the observed responses. This logisticrandom-effect model includes a random intercept to account for thevariability between subjects. Fixed categorical effects includestratification factor, treatment, visit, and treatment-by-visitinteraction, as well as the continuous fixed covariates of baselinescore. (3) A sensitivity analysis was performed by adjusting forbaseline CDAI score and/or other baseline covariates by extending themodel planned above for subjects in the PP Population.

Secondary Endpoints included:

1) CDAI remission at Week 8, as defined by a CDAI score of <150;

2) A reduction of at least 100 points from baseline in CDAI at Week 8;

3) A reduction of at least 70 points from baseline in CDAI at Week 8;

4) CDAI response (either remission defined by CDAI<150 or a CDAIreduction from baseline of at least 100 points from baseline) at Week12. Secondary endpoints 1, 2, 3, and 4 were analyzed in a similar way tothe primary endpoint. In addition, a sensitivity analysis was performedby carrying forward the last response on or before the Week 8 visit toWeek 12 for subjects who had increased steroid (defined as 5 mg/dayprednisolone, or equivalent, or 3 mg/day budesonide).5) Change from baseline CDAI at Week 8. Secondary endpoint 5 wasanalyzed by using the inverse probability weighting generalizedestimating equations method adjusting for prior anti-TNFα use. Also asensitivity analysis was performed using an ANCOVA model after missingdata was imputed using the LOCF approach through the end of thedouble-blind, placebo-controlled, treatment period adjusting for prioranti-TNFα use.6) To evaluate the safety and tolerability of MEDI2070, the safety andtolerability endpoints included AEs including SAEs, significant changesin laboratory values and vital signs. All safety-related endpoints forthe 12-week, double-blind, placebo-controlled, treatment period werereported based on safety population using the actual treatment received.Subjects randomized to placebo with at least one dose of MEDI2070 wereincluded in the active arm; also the subjects randomized to the MEDI2070arm without any active dose were included in the placebo arm. TheMedical Dictionary for Regulatory Activities (MedDRA) was used to codeall AEs. Treatment-emergent AEs were defined as any AE with onset on orafter the administration of the first dose of investigational product upto and including 36 weeks post-last dose.7) To evaluate the PK and immunogenicity (IM) of Multiple Doses ofMEDI2070, descriptive statistics of serum MEDI2070 concentration datawere provided by visit. Individual and mean serum concentration-timeprofiles of MEDI2070 were generated For PK data analysis, time zero wasdefined as the beginning of infusion. The presence of anti-drugantibodies (ADAs) to MEDI2070 in serum was also assessed.

Example 2 Phase 2a Clinical Study Results

I. Study Design and Patients

The phase 2a study (clinicaltrials.gov identifier: NCT01714726)described in Example 1 included a 12-week, double-blind,placebo-controlled treatment period followed by a 100-week open-labeltreatment period to evaluate short-term efficacy and short- andlong-term safety of MEDI2070 in patients with active moderate to severeCrohn's disease who failed prior anti-TNF-α therapy. Conducted at 60centers worldwide, the study included adults aged 18 to 65 yearsdiagnosed with ileal, ileo-colonic, or colonic Crohn's disease for atleast 6 months.

Patients had to have moderate to severe active Crohn's disease (definedas a Crohn's Disease Activity Index [CDAI] score of 220-450), withevidence of active inflammation (baseline C-reactive protein [CRP]≥5mg/L, fecal calprotectin [FCP]≥250 μg/g, or endoscopic evidence ofinflammation [photographic documentation of at least 3 nonanastomoticulcerations, each >0.5 cm in diameter, or 10 aphthous ulcerationsinvolving at least 10 cm of contiguous intestine] within 12 weeks beforescreening). Patients must have received at least one anti-TNF-α therapyat approved doses for Crohn's disease, with primary nonresponse (signsand symptoms of active disease, despite at least 1 induction regimen,including at least 2 doses of anti-TNF-α therapy at least 2 weeks apart)or secondary nonresponse (recurrence of symptoms of persistently activedisease during maintenance anti-TNF-α therapy following initial clinicalbenefit) or intolerance (including but not limited to infusion-relatedreaction, demyelination, congestive heart failure, or infection).Exclusion criteria included having an allogeneic bone marrow transplantor cell-based transplantation; short-bowel syndrome; obstructivestricture within 3 months of study; bowel surgery within 12 weeks ofstudy; ileostomy or colostomy; a clinically significant concomitantdisease; or prior treatment with a biologic agent targeting IL12 orIL23.

Washout of other prior therapies was required, including infliximab for8 weeks before study, adalimumab or certolizumab for 10 weeks,natalizumab for 12 weeks; cyclosporine, mycophenolate mofetil,sirolimus, thalidomide, or tacrolimus for 4 weeks; intravenous orintramuscular corticosteroids for 2 weeks; or topical mesalamine orrectal corticosteroids for 2 weeks. Concomitant use of5-aminosalicylates and glucocorticosteroids was permitted if doses werestable for at least 2 weeks before randomization and remained stablethrough week 8. Similarly, concomitant use of immunomodulators, e.g.,azathioprine, was permitted if the dose remained stable from 8 weeksbefore randomization through week 8. Use of oral antibiotics for Crohn'sdisease, probiotics, and antidiarrheals was allowed.

All patients provided written informed consent. The study was conductedin accordance with the Declaration of Helsinki and Good ClinicalPractice guidelines and each site's local institutional review boardapproved the protocol.

II. Treatment

For the double-blind period, an interactive voice or web-based responsesystem was used for randomization to treatment arms, assigning uniquerandomization codes to each patient. Randomization was stratified basedon the number of prior failed anti-TNF-α agents (1 vs >1). Using blindedrandomization with concealed allocation based on a permutation blockalgorithm, patients were randomized 1:1 within each stratum to receiveMEDI2070 700 mg or placebo intravenously over 60 minutes at week 0(day 1) and week 4 (day 29). Patients, investigators, and the sponsorwere blinded to treatment until the last patient reached week 12, whenthe primary analysis was conducted. During the open-label period (weeks12 through 112), all patients received MEDI2070 210 mg subcutaneouslyevery 4 weeks (26 doses over 100 weeks).

III. Study Assessments

The primary outcome measure was the proportion of patients achievingclinical effect, defined as at least a 100-point CDAI score reductionfrom baseline or CDAI score less than 150 at week 8. Secondary measuresincluded the proportion of patients achieving CDAI remission (defined asCDAI score <150) at week 8, at least a 100-point CDAI score reduction(CR100) from baseline at week 8, at least a 70-point CDAI scorereduction (CR70) from baseline at week 8, clinical effect at week 12,CDAI remission at week 12, and the safety and tolerability of MEDI2070,including adverse events, serious adverse events, and significantchanges in laboratory values and vital signs. Serum MEDI2070concentrations and the presence of ADAs were evaluated at baseline, week8, week 24, and end of study.

Exploratory outcome measures included inflammatory markers in the bloodand stool (CRP and FCP), responses of other biomarkers and theirpredictive value for clinical effects, sustained clinical effect betweenweeks 8 and 24, and sustained CDAI remission assessed at weeks 8 and 24.Biomarker assessments included changes in IL22 serum levels (expressedin high levels in Crohn's disease patients and a marker of diseaseactivity [Schmechel et al., Inflamm Bowel Dis, 14:204-212 (2008)]) frombaseline after treatment with MEDI2070 versus placebo at weeks 8 and 12;association of change from baseline in IL22 levels with clinical effectand CDAI remission after treatment with MEDI2070 versus placebo at weeks8 and 12; and assessment of higher IL22 serum levels at baseline as apredictor of clinical effect and CDAI remission after treatment withMEDI2070 compared with placebo at weeks 8 and 12.

IV. Statistical Analysis

Assuming a CDAI clinical effect rate of 20% in the placebo group,approximately 54 patients per treatment arm were required to provide 87%power to detect a 25% difference in CDAI clinical effect rates at week 8between MEDI2070 700 mg and placebo, using a two-sided test atsignificance level of a=0.1. Assuming an approximately 10% dropout rateper treatment arm adjustment, approximately 60 patients were to berandomized.

The modified intent-to-treat population included all randomized patientswho received at least one dose of study treatment during thedouble-blind period. The safety population comprised patients whoreceived any study treatment during the double-blind period. Theper-protocol population included patients who received all treatmentdoses and had no major protocol deviations. The open-label populationincluded all patients who received at least 1 dose of MEDI2070 duringthe open-label period.

For the primary and secondary measures with binary outcomes, comparisonbetween treatment arms was based on the modified intent-to-treatpopulation, using a logistic regression model [Ge et al., Drug Inf445:481-493 (2011)], with treatment and stratification factor (number ofprior anti-TNF-α agents of 1 vs >1) as covariates. The significance ofthe treatment effect was tested using the 2-sided test at α=10%. Asensitivity analysis was conducted by extending the planned logisticregression model to adjust for baseline CDAI score and/or other baselinecovariates. Missing data for dichotomous end points were imputed usingnon-responder imputation. A patient imputed as a nonresponder beforeweek 8 was considered a nonresponder for all subsequent visits.Additionally, patients with a clinically meaningful increase in steroiduse were assumed to be nonresponders. Clinically meaningful increase insteroid dose was defined as an increase of at least 5 mg/day for atleast 3 days of prednisone or equivalent, or an increase of at least 3mg/day for at least 3 days of budesonide. Missing data for continuousmeasures were handled using the inverse probability weightinggeneralized estimating equations method, adjusting for prior anti-TNF-αagent use.

Pharmacokinetic data were summarized. Exploratory analyses wereperformed for patients in the open-label period. Efficacy data from thedouble-blind and open-label periods up to week 24 for these patientswere combined and reported by treatment arms in the double-blind period.Change from baseline in CRP and FCP were analyzed using a mixed-effectsrepeated measures model, adjusting for prior anti-TNF-α use andbaseline, for the open-label population. Comparisons between MEDI2070210 mg at week 24 and MEDI2070 700 mg or placebo at week 12 wereperformed.

V. Patient Results

In total, 121 patients were randomized; 119 of these receiveddouble-blind treatment (FIG. 1). In the double-blind period, 52 of 59(86.7%) and 52 of 60 (85.2%) patients completed week 12 in the MEDI2070and placebo groups, respectively. Baseline patient characteristicsgenerally were balanced between treatment groups (TABLE 1), except CDAIand CRP levels were numerically higher in the MEDI2070 group. Resultsfrom weeks 8, 12, and 24 are reported here; the study is ongoing forlong-term follow-up.

TABLE 1 Baseline Characteristics and Demographics (modifiedintent-to-treat population). MEDI2070 Placebo Total Parameter (N = 59)(N = 60) (N = 119) Mean age, yr (SD) 34.9 (11.2) 38.1 (10.7) 36.5 (11.0)Female, no. (%) 37 (62.7) 37 (61.7) 74 (62.2) Mean weight, kg (SD) 70.4(20.7) 71.9 (15.4) 71.2 (18.2) Mean disease duration, 13.2 (9.4) 11.2(8.5) 12.2 (9.0) yr (SD) ≥2 yr, % 96.6 90.0 93.3 Sites of disease, no.(%) Ileal only 14 (23.7) 18 (30.0) 32 (26.9) Colonic only 16 (27.1) 18(30.0) 34 (28.6) Ileo-colonic 28 (47.5) 24 (40.0) 52 (43.7) Other 1(1.7) 0  1 (0.8) Crohn's Disease Activity Index score Mean (SD) 325.0(59.2) 312.4 (56.3) 318.6 (57.8) Minimum, maximum 222, 440 221, 450 221,450 Mean C-reactive protein, 29.8 (35.4) 21.1 (24.2) 25.4 (30.4) mg/L(SD) Mean C-reactive protein 46 (78.0) 39 (65.0) 85 (71.4) ≥5 mg/L, no.(%) Mean fecal calprotectin, 536.7 (303.2) 616.9 (420.7) 578.2 (369.3)μg/g (SD)* Mean fecal calprotectin 41 (73.2) 47 (78.3) 88 (75.9) ≥250μg/g, no. (%) Prior anti-tumor necrosis factor-α agents, no. (%) 1 18(30.5) 19 (31.7) 37 (31.1) 2 35 (59.3) 35 (58.3) 70 (58.8) ≥3 6 (10.2) 6(10.0) 12 (10.1) Prior use of anti-tumor necrosis factor-α agents, no.(%) Infliximab 51 (86.4) 50 (83.3) 101 (84.9) Adalimumab 45 (76.3) 45(75.0) 90 (75.6) Certolizumab 10 (16.9) 11 (18.3) 21 (17.6) Reason fordiscontinuing prior anti-tumor necrosis factor-α agent, no. (%) Primaryfailure 23 (39.0) 23 (38.3) 46 (38.7) Secondary failure 34 (57.6) 33(55.0) 67 (56.3) Intolerance 27 (45.8) 26 (43.3) 53 (44.5) Other 7(11.9) 11 (18.3) 18 (15.1) Not applicable 0  2 (3.3) 2 (1.7)5-aminosalicylate use 18 (30.5) 18 (30.0) 36 (30.3) at baseline, no. (%)Corticosteroid use at 24 (40.7) 24 (40.0) 48 (40.3) baseline, no. (%)Immunomodulators use 18 (30.5) 14 (23.3) 32 (26.9) at baseline, no. (%)Prior surgery for 29 (49.2) 26 (43.3) 55 (46.2) Crohn's disease, no. (%)*Three patients had missing fecal calprotectin assessments.VI. Efficacy(i) Week 8

For the primary outcome measure, the rate of clinical effect at week 8was significantly higher in the MEDI2070 group versus the placebo group(49.2% vs. 26.7%, respectively; P=0.01; point estimate: 0.225 [90%confidence interval (CI): 0.083 to 0.368]; FIG. 2A). CDAI remissionrates were 27.1% with MEDI2070 and 15.0% with placebo (P=0.10; pointestimate: 0.122 [90% CI: 0.000 to 0.243]). CR70 rates were 52.5% and46.7% in the MEDI2070 and placebo groups, respectively (P=0.52), andCR100 rates were 45.8% and 25.0% in the MEDI2070 and placebo groups,respectively (P=0.02).

A significantly greater proportion of patients receiving MEDI2070achieved the composite end points of CDAI effect and 50% reduction inFCP or CRP versus baseline (P<0.001), and CDAI remission and 50%reduction in FCP or CRP versus baseline (P=0.02; FIG. 2B). MEDI2070treatment also resulted in significantly greater reductions in FCPversus placebo (least squares mean change −153.5 vs. −49.7; leastsquares mean difference −105.6 [90% CI: −184.0 to −27.2]; P=0.027) andsignificantly reduced CRP levels (least squares mean change: −12.6 vs.5.1 with placebo; least squares mean difference −17.6 [90% CI: −28.3 to−6.9]; P=0.007).

(ii) Week 12

At week 12, the rate of clinical effect was greater with MEDI2070 (37.3%vs 28.3%; P=0.29), as was the rate of CDAI remission (20.3% vs. 13.3%;P=0.31); however, the differences were not statistically significant.Rates of CR100 at week 12 were 37.3% with MEDI2070 and 28.3% withplacebo (P=0.288). At week 12, significant reductions in FCP and CRPwith MEDI2070 were maintained (FCP: least squares mean change −179.6 vs−55.1; least squares mean difference −124.6 [90% CI: −221.2 to −27.9];P=0.034; CRP: least squares mean change: −13.4 vs −2.6 with placebo;least squares mean difference −10.8 [90% CI: −17.6 to −4.1]; P=0.008).

(iii) Week 24

Clinical effect and CDAI remission rates were maintained in the MEDI2070group at 24 weeks. The proportion of patients receiving placebo thenopen-label MEDI2070 (placebo/MEDI2070 group) who achieved clinicaleffect and CDAI remission was similar to those receiving MEDI2070 700 mgthen open-label MEDI2070 (MEDI2070/MEDI2070) at week 24 (FIG. 3A andFIG. 3B). Over the 24-week period, the MEDI2070/MEDI2070 group continuedto achieve the composite end points of CDAI effect plus 50% reduction inFCP or CRP versus baseline, and CDAI remission plus 50% reduction in FCPor CRP versus baseline (TABLE 2). The proportion of patients in theplacebo/MEDI2070 group achieving both composite end points at week 24was similar to those in the MEDI2070/MEDI2070 group. In theMEDI2070/MEDI2070 group, changes from baseline in FCP and CRP levelswere maintained between weeks 12 and 24; in the placebo/MEDI2070 group,decreases in FCP and CRP levels were observed between weeks 12 and 24.

TABLE 2 Composite End Points (weeks 8 through 24), Sustained Effect andRemission (open-label population), and Change from Baseline in FecalCalprotectin and C-reactive Protein (mixed-effects model; open-labelpopulation). MEDI2070 700 mg/ Placebo/ MEDI2070 210 MEDI2070 210Parameter, no. (%) mg (N = 52) mg (N = 52) Crohn's Disease Activityindex effect and ≥50% reduction in fecal calprotectin or C-reactiveprotein vs baseline (nonresponder imputation) Week 8 24 (46.2) 6 (11.5)Week 12 22 (42.3) 5 (9.6) Week 24 24 (46.2) 25 (48.1) Crohn's DiseaseActivity index remission and ≥50% reduction in fecal calprotectin orC-reactive protein vs. baseline (nonresponder imputation) Week 8 14(26.9) 5 (9.6) Week 12 12 (23.1) 4 (7.7) Week 24 19 (36.5) 18 (34.6)Sustained effect at weeks 22 (42.3) 12 (23.1) 8 and 24* (nonresponderimputation) Sustained remission at 12 (23.1) 6 (11.5) weeks 8 and 24^(†)(nonresponder imputation) Mean change in fecal calprotectin, (μg/g)^(‡)Double-blind period −186.5 (n = 40) −46.0 (n = 41) (Week 12) Open-labelperiod −252.3 (n = 36) −253.8 (n = 39) (Week 24) Difference at week 12−65.7 (−161.0 to 29.5) −207.8 (−307.0 to −108.5) vs week 24 (90% CI) Pvalue 0.254 <0.001 Mean change in C reactive protein (mg/L)^(‡)Double-blind period −16.4 (n = 51) 0.1 (n = 52) (Week 12) Open-labelperiod −18.2 (n = 45) −10.6 (n = 49) (Week 24) Difference at Week 12−1.8 (−7.2 to 3.6) −10.8 (−16.4 to −5.1) vs Week 24 (90% CI) P value0.580  0.002 *Sustained effect is defined as achieving the criteria ofeffect at both week 8 and week 24. Percentage calculated using number ofpatients in the open-label population. ^(†)Sustained remission isdefined as achieving the criteria of remission at both week 8 and week24. Percentage calculated using the number of patients in the open-labelpopulation. ^(‡)Mean and P value were obtained from a mixed-effectsmodel with fixed terms for treatment group, visit, treatment by visitinteraction, stratification factor of prior anti-tumor necrosis factor-αuse, and baseline fecal calprotectin or C-reactive protein, and patientswithin treatment as random effect, assuming unstructured covariancestructure.VII. MEDI2070 Exposure-Response Relationship

At the 700-mg intravenous dose of MEDI2070, no definitive relationshipbetween exposure and efficacy was established (data not shown). SerumMEDI2070 concentrations at the week 4 trough and at weeks 8 and 12varied over a tenfold range and were not related to CDAI response ornonresponse at week 8 (data not shown). These findings are consistentwith dosing at the plateau of the dose-response curve.

VIII. Biomarkers

Patients in the MEDI2070 group had greater reductions in serum IL22levels versus those in the placebo/MEDI2070 group. Baseline serum IL22levels greater than or equal to the median value of 15.6 pg/mL wereassociated with an increased likelihood of clinical effect in theMEDI2070 group. Patients in the MEDI2070 group with baseline IL22 levelsless than 15.6 pg/mL had a CR100 response rate similar to that in theplacebo group (FIG. 5). Clinical response was not a strong function ofIL22 in the placebo group.

IX. Safety

(i) Week 12

At the end of the double-blind period, the proportion of patients withtreatment-emergent adverse events was similar between the MEDI2070 andplacebo groups (67.8% vs. 68.3%, respectively), as were the proportionof patients with grade 3 or greater adverse events (10.2% vs. 11.7%,respectively) and serious adverse events (8.5% vs. 8.3%, respectively).Treatment-related adverse events were observed in 13.6% of patientsreceiving MEDI2070 and 21.7% of patients receiving placebo; thoseobserved in at least 5% of patients in either group are shown in TABLE3. Serious adverse events occurred in five patients in the MEDI2070group and five in the placebo group; events included Crohn's disease(three events in two patients), colonoscopy-associated colon perforation(n=1), pyrexia (n=1), and cellulitis (n=1) in the MEDI2070 group, andanemia (n=1), Crohn's disease (two events in two patients), diarrhea(n=1), gastrointestinal hemorrhage (n=1), and abdominal abscess (n=1) inthe placebo group.

TABLE 3 Treatment-Emergent Adverse Events in at Least 5% of Patients inEither Treatment Group (up to week 12; safety population) Adverse Event,n (%) MEDI2070 (N = 59) Placebo (N = 60) Headache 10 (16.9) 4 (6.7)Nasopharyngitis 8 (13.6) 6 (10.0) Abdominal pain 6 (10.2) 6 (10.0)Crohn's disease 5 (8.5) 5 (8.3) Vomiting 3 (5.1) 2 (3.3) Arthralgia 3(5.1) 2 (3.3) Proctalgia 3 (5.0) 0 Dizziness 3 (5.0) 0 Pyrexia 2 (3.4) 4(6.7) Nausea 2 (3.4) 3 (5.0) Diarrhea 0 5 (8.3) Sinusitis 0 4 (6.7)Insomnia 0 3 (5.0) Cough 0 3 (5.0)

Infections that were serious or at least grade 3 in severity, or thatrequired oral or parenteral antimicrobial therapy, occurred in fourpatients with four events in the MEDI2070 group, and in seven patientswith 11 events in the placebo group. Treatment discontinuationattributable to treatment-emergent adverse events occurred in 8.5% ofMEDI2070 patients (including gastrointestinal disorders [6.8%] andinfection [1.7%]) and in 10.0% of placebo patients (includinggastrointestinal disorders [6.7%], infection [1.7%], and eye disorder[1.7%]).

(ii) Week 24

At week 24, treatment-emergent adverse events were observed in 67.3% ofpatients in the MEDI2070 group and 65.4% of those in theplacebo/MEDI2070 group, with treatment-related adverse events observedin 25.0% and 21.2% of patients, respectively. Treatment-emergent adverseevents of at least grade 3 severity occurred in 13.5% of patients in theMEDI2070 group and 3.8% of patients in the placebo/MEDI2070 group;serious adverse events were observed in 15.4% and 7.7% of patients,respectively. Treatment discontinuation attributable totreatment-emergent adverse events occurred in 9.6% of MEDI2070 patients(including Crohn's disease flare [3.8%], anal fistula [1.9%], anemia andlymphopenia [1.9%], and anal abscess [1.9%]), and in 3.8% ofplacebo/MEDI2070 patients (including pelvic abscess [1.9%] and kidneystones [1.9%]). The number of infection events that were serious, atleast grade 3 in severity, or required oral or parenteral antimicrobialtherapy were equal in the MEDI2070 and placebo/MEDI2070 groups (13events).

X. Immunogenicity

Antidrug antibodies were detected in two of 119 patients. One patientreceiving MEDI2070 had antidrug antibodies at baseline, but not insubsequent assessments. The other patient, who received placebo duringthe double-blind period, had antidrug antibodies at week 24.

XI. Discussion

In patients with moderate to severe Crohn's disease who failed prioranti-TNF-α therapy, MEDI2070 resulted in a significantly greater rate ofclinical effect, a composite of a 100-point reduction in CDAI and CDAIremission, at 8 weeks compared with placebo (49.2% vs. 26.7%,respectively; P=0.01), meeting the primary study end point. Clinicalefficacy was consistent with the biologic effects observed withMEDI2070. Patients in the MEDI2070 group had greater decreases frombaseline in FCP and CRP relative to placebo. The beneficial effects ofMEDI2070 on FCP and CRP are remarkable given that this patientpopulation was previously heavily treated with anti-TNF-α therapies;more than 65% of patients had received two or more prior anti-TNF-αagents.

At week 12, the rate of clinical effect was greater in the MEDI2070group, as was the rate of CDAI remission; however, these differenceswere not significant. Significant reductions in FCP and CRP withMEDI2070 were maintained at week 12. At week 24, clinical effect, CDAIremission, and changes from baseline in levels of FCP and CRP weremaintained between weeks 12 and 24 in the MEDI2070 group. Among patientsin the placebo/MEDI2070 group, the proportion achieving clinical effectand CDAI remission at week 24 was similar to those who received MEDI2070in both periods, and significant decreases in FCP and CRP levels wereobserved between weeks 12 and 24. At the 700-mg intravenous dose ofMEDI2070, no definitive relationship between exposure and efficacy wasestablished. This finding is consistent with dosing at the plateau ofthe dose-response curve. A wider range of doses will be explored infuture studies.

MEDI2070 was well tolerated, with rates of treatment-emergent adverseevents, grade 3 or greater adverse events, serious adverse events, anddiscontinuations owing to treatment-emergent adverse events similar tothose of placebo at 12 weeks. Rates of serious or severe infections wereless than 10% in the MEDI2070 group at week 12. At week 24, overallrates of treatment-emergent adverse events were similar between theMEDI2070 and placebo/MEDI2070 groups; rates of grade 3 or greateradverse events and serious adverse events were numerically greater inthe MEDI2070 group versus the placebo/MEDI2070 group (13.5% vs. 3.8% and15.4% vs. 7.7%, respectively), as were rates of discontinuationsattributable to treatment-emergent adverse events (9.6% vs. 3.8%,respectively). Rates of serious or severe infections were the same inboth treatment groups at 24 weeks (13 events). One patient had antidrugantibodies at baseline; presumably, this was a false-positive result andwas not detected on follow-up assessment.

Although specific inclusion criteria, primary end points, and timing ofassessments vary, the results of our study suggest that the short-termCDAI remission rate achieved with MEDI2070 (27.1% at 8 weeks) generallycompares favorably with those of other biologic therapies evaluated inpatients with Crohn's disease who failed prior anti-TNF-α therapy. Theseinclude ustekinumab (26% [7/27] at 8 weeks) (Sandborn et al.Gastroenterology 135:1130-41 (2008)), vedolizumab (10.5% [n=105] at 6weeks), (Sandborn et al. N Engl J Med 369:711-21 (2013)) and adalimumab(21% [34/159 with secondary nonresponse to infliximab] at 4 weeks)(Sandborn et al. Ann Intern Med 146:829-38 (2007)). The safety profileof MEDI2070 also compares favorably with other biologics available forpatients with Crohn's disease. A recent meta-analysis of studiesevaluating the dual IL23/IL12 inhibitors ustekinumab and briakinumabfound an increased risk of major adverse cardiovascular events inpsoriasis patients treated for 12 to 20 weeks [Tzellos et al., J EurAcad Dermatol Venereol, 27:1586-1587 (2013)]. No patients in our studyexperienced a major adverse cardiovascular event up to 24 weeks offollow-up. MEDI2070 is specific for IL23 and does not inhibit IL12; theclinical significance of this specificity is not known. However,follow-up for this study (104 weeks) is ongoing to determine longer-termefficacy and safety.

We evaluated serum IL22 levels at baseline and following MEDI2070treatment. IL22, expressed at high levels in Crohn's disease, is aneffector cytokine that supports mucosal barrier integrity and is anindicator of IL23 axis activity [Schmechel et al., Inflamm Bowel Dis,14:204-212 (2008)]. Serum IL22 levels were reduced in the MEDI2070 groupcompared with the placebo group. Additionally, baseline serum IL22levels greater than or equal to 15.6 pg/mL were associated with anincreased likelihood of clinical effect in the MEDI2070 group, whereasMEDI2070-treated patients with baseline IL22 levels less than 15.6 pg/mLhad CR100 responses similar to those in the placebo group. FIG. 5. Incontrast, in a study by Dige et al., IL22 levels were not reduced inCrohn's disease patients effectively treated with adalimumab [Dige etal., J Crohns Colitis, 7:248-255 (2013)]. This study is among the firstto incorporate novel biomarkers to further the understanding of thepathogenesis and improve the treatment of Crohn's disease.

In conclusion, MEDI2070 treatment demonstrated consistently robustefficacy, with an acceptable safety profile in patients with Crohn'sdisease who failed prior anti-TNF-α therapies.

Example 3 ELISA Immunoassay for Detection and Quantification of CCL20

CCL20 can be detected and quantified according to the exemplary methoddisclosed in this Example (Example 3). This method was applied, forexample, to obtain the CCL20-related experimental data presented inExample 5 (see below). The method was also used to test samples for thephase 2a study presented in Examples 1 and 2.

CCL20 (MIP3 alpha) was measured using a quantitative ELISA basedimmunoassay. A Human CCL20/MIP-3a Quantikine Elisa kit from R&D Systems(Cat # DM3A00) was used. The kit included a 96 well plate pre-coatedwith capture antibody and pre-blocked (Part #890831) ready for theapplication of 1000 per well, of either Reference standards (RS),quality controls (QC), negative control (NC), or serum test samplesdiluted to the Minimum Required Dilution (MRD) of 1:4 using the providedRD6U calibrator diluent (Part #895148).

The assay plate was incubated for 2 hours±0.5 hours at room temperature(RT) on a plate shaker with gentle shaking and unbound analyte wasremoved by washing the plate 4 times with Medimmune Elisa-wash buffer(1×PBS pH 7.4/0.05% Tween-20). To detect bound analyte, 200 μlofanti-MIP-3alpha conjugate (Part #890832) was added to each well and theplate was incubated for an additional 2 hours±0.5 hours at roomtemperature (RT) on a plate shaker with gentle shaking. Unbounddetection conjugate was removed by washing the plate 4 times with theWash buffer (1×PBS pH 7.4/0.05% Tween-20).

To develop the plate, 200 μl of Substrate Solution (1:1 Mixed Parts#895000 & 895001) was then added to each well and the plate wasincubated for 30 minutes at room temperature protected from light andthen 50 μl of Stop Solution (Part #895032) was added to each well. Theplate was gently tapped to mix thoroughly and the optical density of 450nM and 570 nM was determined using a SpectraMaxPlus MicroplateSpectrophotometer (Molecular Devices) within 30 minutes. The CCL20concentrations in samples and controls were interpolated from thequadratic curve fitting of the standard curve of recombinant CCL20provided in the kit and run on each plate. The assay was determined tobe specific, reproducible and precise with a quantifiable range of 4pg/ml-2 ng/ml of CCL20 in 100% serum.

Example 4 Immunoassays for Detection and Quantification of IL23 PathwayBiomarkers IL22 and LCN2

IL22 and/or LCN2 can be detected and quantified according to theexemplary methods disclosed in this Example (Example 4). These methodswere applied, for example, to obtain the experimental data presented inExample 5 (see below). The methods were also used to test samples forthe phase 2a study presented in Examples 1 and 2.

3.1 IL22 ELISA Immunoassay

IL22 levels were measured using a quantitative ELISA-based immunoassay.A mouse monoclonal antibody specific for human IL-22 was pre-coated ontoa microplate (R&D Systems, Cat # D2200). One hundred microliters ofassay diluent were first added to wells of microplates followed byaddition of 100 μL of standards, controls and samples. The plates wereincubated for 2 hours±15 minutes at room temperature to allow IL22 tobind to the capture antibody on the plates. Plates were then washed 5times using 1× Wash buffer (1×PBS pH 7.4/0.05% Tween-20) to removeunbound materials and were further incubated with 200 μL of thedetection antibody (anti-IL22 antibody-HRP conjugate, (R&D Systems, Cat# D2200) for 2 hours±15 minutes at room temperature. After that, theplates were washed again and incubated with 150 μL of TMB, a chromogenicHRP substrate (Neogen, Cat #331176) for 20 minutes±3 minutes at roomtemperature in the dark. The enzyme reaction was stopped by the additionof 100 μL of stop solution (1M HCL).

Within 30 minutes after stopping the reaction, plates were read on aSpectraMaxPlus 384 Microplate Spectrophotometer (Molecular Devices) tomeasure the optical density at 450 nm. The intensity of the colorgenerated is directly proportional to the amount of bound IL22. The IL22concentrations in samples and controls were interpolated from thestandard curve of recombinant E. coli-derived IL22, which was run oneach plate. The quadratic model was used for curve fitting. The assaywas reproducible and precise. The quantifiable range was established tobe 10 pg/mL-800 pg/mL of IL-22 in 100% serum.

3.2 LCN2 ELISA Immunoassay

A standard Meso Scale Discovery plate (MSD, Cat # L15XA) was coated with1 μg/mL of a rat-anti-human Lipocalin-2 antibody (R&D Systems, Cat #MAB17571), at 50 μL/well at 2° C.-8° C. overnight. The plate was washedthree times with 200 μL ELISA Wash Buffer (0.05% Tween-20/PBS) andblocked with 150 μL/well of I-Block buffer (IBB) (0.5% Tween-20/0.2%I-Block Buffer/PBS) for at least 60 minutes on a plate shaker withgentle shaking. Reference standards, quality controls and negativecontrol, prepared in IBB, and serum test samples, diluted to the minimumrequired dilution of 1:50 in IBB, were added to the plate at 30 μL/well.

The assay plate was incubated for 2 hours at room temperature on a plateshaker with gentle shaking. Unbound analyte was removed by washing theplate. To detect bound analyte, 0.5 μg/mL of a biotin-conjugatedgoat-anti-human Lipocalin-2 antibody (R&D Systems, Cat # BAF1757) wasadded at 30 μL/well and the plate was incubated for an additional 60minutes on a plate shaker with gentle shaking. Unbound detectionantibody was removed by washing the plate. Streptavidin SULFO-TAG™detection dye (MSD, Cat # R32AD-1) was added to the plate at 0.5 μg/welland the plate was incubated for an additional 30 minutes covered toprotect from light exposure. The plate was washed and 1× Read Buffer(MSD, Cat # R92TC-2) was added at 150 μL/well and the plate was read ona MSD Sector Imager within 20 minutes.

ECL (electrochemiluminescence) values for each plate were collectedusing the MSD Sector Imager. The ECL values for the reference standardswere plotted with Softmax Pro GxP v6.4 software (Molecular Devices,Sunnyvale, Calif.) using 5-Parameter Logistic model of curve fitting.The concentrations of unknown samples were interpolated from thestandard curves on the same assay plate. The Softmax-derived data wasthen imported into Microsoft Excel Software to generate data reports andgraphs. The assay's dynamic range was established to be from 0.70 ng/mlto 1000.00 ng/mL adjusted to 100% serum.

Example 5 Identification of IL23 Pathway Biomarkers (CCL20, IL22 and/orLCN2) as Predictive Biomarkers for Treatment of IL23-Mediated Diseaseswith an Anti-IL23 Antibody

IL23 is expressed primarily from activated dendritic cells andmacrophages (see Gaffen et al (2014) Nature Revs Immunol 14: 585-600;Oppmann et al (2000) Immunity 13: 715-251) and acts directly on avariety of hematopoetic cell types including Th17, Th22, δδ T cells andinnate lymphoid cells (ILCs) to induce cytokines including IL22, IL21,IL17A, IL17F, IL17A/F, TNF alpha and GM-CSF (see, e.g., Gaffen et al(2014) Nature Revs Immunol 14: 585-600; Zheng et al (2007) Nature 445:648-51; El-Behi et al (2011) Nature Immunol 12: 568-575). These effectorand regulatory cytokines can in turn act on a variety other cell typesexpressing the appropriate cognate receptors. IL23-induced IL22, forexample, can stimulate IL22-receptor expressing epithelial cells andkeratinocytes to secrete antimicrobial proteins such as LCN2 (Sonnenberget al (2010) Adv Immunol 107: 1-29; Stallhofer et al (2015) InflammBowel Dis 2015 Aug. 7; Behnsen et al (2014) Immunity 40: 262-73).

In the Phase 2a study described in Examples 1 and 2, CD patients withelevated baseline serum IL22 or LCN2 levels of greater than or equal to15.6 pg/mL or 215 ng/mL, respectively (as measured using theimmunoassays described in Example 3), had an increased likelihood ofclinical effect in the MEDI2070 group, whereas MEDI2070-treated patientswith baseline IL22 levels less than 15.6 pg/mL had CR100 responsessimilar to those in the placebo group, and those patients with baselineLCN2 levels less than 215 ng/mL did not show statistically significantincreased CR100 responses compared to placebo. FIGS. 5 and 6. Inparticular, patients with baseline serum IL22 levels≥15.6 pg/mL orpatients with baseline serum LCN2 levels≥215 ng/mL were observed to havestatistically significant increased CDAI-100 responses when treated withMEDI2070 compared to placebo at week 8. FIGS. 5 and 6. Similarly, wealso observed that CD patients with reduced baseline serum CCL20 levelsof less than 22.6 pg/mL were associated with an increased likelihood ofclinical effect in the MEDI2070 group, whereas MEDI2070-treated patientswith baseline CCL20 levels greater than or equal to 22.6 pg/mL did notshow statistically significant increased responses compared to placebo.FIG. 4. Thus as shown in FIGS. 4-6, patients with baseline serum IL22levels≥15.6 pg/mL, patients with baseline serum LCN2 levels≥215 ng/mL orpatients with baseline serum CCL20 level<22.6 pg/mL all were observed tohave statistically significant increased CDAI-100 responses when treatedwith MEDI2070 compared to placebo.

To further understand the relationship between baseline serum IL22, LCN2and/or CCL20 levels and response to treatment with MEDI2070, the set ofbaseline values of either IL22, LCN2 or CCL20 across the entire studypopulation was divided into 10 levels, or deciles, such that each of the11 analyte cut-offs progressively segmented the study population intogroups with 10% less of the total study population. The differentialclinical response rate between MEDI2070 and placebo exposed subjects asa function of baseline IL22, LCN2, and CCL20 serum levels at each decilecut-off is provided in FIGS. 7-9 and the individual IL22, LCN2, andCCL20 serum decile values is summarized below in TABLE 4 (IL22), TABLE 5(LCN2), and TABLE 6 (CCL20). For example, as reported in TABLE 4, at the4^(th) decile, 40% of the total study population had a baseline IL-22level of <12.7 pg/mL and 60% of the total study population had abaseline IL-22 level≥12.7 pg/mL. The CDAI response rate at week 8 (asmeasured by the percentage (%) of subjects achieving a CDAI score <150or a reduction in CDAI score of >100) in those subjects exposed toMEDI2070 and with baseline IL-22 levels≥12.7 pg/mL, i.e. above the4^(th) decile, was 58.3%. 21 subjects exposed to MEDI2070 and withbaseline IL22 levels above the 4^(th) decile for the study population(12.7 pg/ml) were found to be CDAI responders at week 8. The function inR called ‘quantiles’ was used to determine decile values. As anotherexample, as reported in TABLE 6, at the 4^(th) decile, 40% of the totalstudy population had a baseline CCL20 level of <20.8 pg/mL and 60% ofthe total study population had a baseline CCL20 level≥20.8 pg/mL. TheCDAI response rate at week 8 (as measured by the percentage (%) ofsubjects achieving a CDAI score <150 or a reduction in CDAI scoreof >100) in those subjects exposed to MEDI2070 and with baseline CCL20levels<20.8 pg/mL, i.e., below the 4th decile, was 51.6%. 16 subjectsexposed to MEDI2070 and with baseline CCL20 levels below the 4^(th)decile for the study population (20.8 pg/mL) were found to be CDAIresponders at week 8. The numbers of CDAI responder and non-respondersin the MEDI2070 and placebo exposed groups, and the CDAI response ratesat week 8 of the study in subjects with baseline IL22, LCN2, or CCL20values greater than or equal to each decile cut are also indicated inTABLES 4-6, and differences between treatment and placebo response ratesfor each decile cut is provided in FIG. 7. Two additional measurementsof clinical response—the difference between the percentage (%) ofsubjects treated with MEDI2070 versus those treated with placeboachieving a 100 point improvement in CDAI score at week 8 (FIG. 8); andthe difference between the percentage (%) of subjects treated withMEDI2070 versus those treated with placebo achieving a CDAI response(CDAI score <150 or a reduction in CDAI score of >100)+also achievinga >50% reduction in either FCP or CRP compared to baseline FCP or CRP atweek 8 (FIG. 9)—as a function of baseline IL22, LCN2, and CCL20levels/deciles described in TABLES 4-6 were also performed.

As shown in FIGS. 7-9 and reported in TABLE 4 below, CD patients treatedwith MEDI2070 having increasingly higher levels of baseline IL22achieved higher CDAI response rates at week 8 compared to placebo (asmeasured using any of the three different clinical response measurementsshown in FIGS. 7-9), illustrating that MEDI2070 induced better clinicalresponses in patients with high baseline IL22 serum levels. Notably,subjects with high levels of IL22 (including, e.g., subjects with IL22levels at the 5^(th), 6^(th) or 7^(th) deciles (0.5, 0.6 or 0.7quantiles)) had greater clinical response rate differences from placebo(irrespective of which of the three different clinical responsemeasurements was used) compared to the IL22 low subjects (including,e.g. subjects with IL22 levels at the 1^(st) or 2^(nd) deciles (0.1 or0.2 quantiles)). See FIGS. 7-9. These IL22 high subjects also hadincreased CDAI response rates compared to all comers treated withMEDI2070 (see, e.g., FIG. 10).

Importantly, the CDAI response rates and CDAI remission rates observedin IL22 high subjects treated with MEDI2070 in the Phase 2a study areamongst the highest clinical response rates to biologics therapy for CDreported to date. For example, as shown in FIG. 10, the CDAI-100response rate differential (defined as the difference in the percentage(%) of subjects achieving a CDAI-100 response between treatment andplacebo) and/or CDAI remission rate differential (defined as thedifference in the percentage (%) of subjects achieving a reduction intotal CDAI score to below 150 points between treatment and placebo)achieved in patients having elevated baseline serum IL22 treated withMEDI2070 for 8 weeks were highly increased compared to the publishedCDAI-100 response and/or CDAI remission rates of patients treated with anumber of other compounds currently approved or under development totreat CD including: Ustekinumab (response rates after 6 weeks or 8 weeksof treatment with a 6 mg/kg dose as reported in FIG. 1 of Sandborn etal., N Engl J Med. 2012 Oct. 18; 367(16):1519-28.); Vedolizumab(response rates after 6 weeks or 10 weeks of treatment as reported inFIG. 3 of Sands et. al., Gastroenterology. 2014 September;147(3):618-627); or Adalimumab (response rates after 4 weeks oftreatment in patients who are secondary failures to infliximab asreported in Sandborn et. al, Ann Intern Med. 2007; 146:829-838). Forexample, both the CDAI-100 response rate differential (“CDAI ResponseDelta vs. Placebo”) and the CDAI remission rate differential (“CDAIRemission Delta vs. Placebo”) achieved in patients treated with MEDI2070for 8 weeks who had a baseline CRP≥5 mg/L; baseline IL-22≥11.3 pg/mL;baseline IL-22≥15.6 pg/mL; or baseline IL-22≥11.3 pg/mL+CRP≥5 mg/L (asmeasured using the IL22 immunoassay described in Example 3) were greaterthan the reported CDAI-100 response rate differential and/or the CDAIremission rate differential for Ustekinumab, Vedolizumab and Adalimumabreported in FIG. 10. The overall clinical response and remission ratesfor all patients treated with MEDI2070 in the Phase 2a study,irrespective of biomarker status, was similar to the response rates ofother molecules currently approved or under development. TABLE 6summarizes the CDAI-100 response rate differential and the CDAIremission rate differential for each of the MEDI2070-treated subgroupsplotted in FIG. 10. These results further underscore the surprising andunexpected predictive value of high or elevated IL22 serum levels (aloneor in combination with other biomarkers disclosed herein) in identifyingpatients having an IL23-mediated disease or disorder responsive totreatment with an IL23 antagonist (including, e.g., an anti-IL23antibody or fragment thereof such as MEDI2070).

Similarly, as shown in FIGS. 7, 8, and 9 and reported in TABLE 5, CDpatients treated with MEDI2070 having increasingly higher levels ofbaseline LCN2 achieved higher clinical response rates (as measured usingany of the three different clinical response measurements shown in FIGS.7, 8 and 9) at week 8 compared to placebo, supporting that MEDI2070induced better clinical responses in patients with high baseline LCN2serum levels. Notably, LCN2 high subjects (including, e.g., subjectswith LCN2 levels at the 5^(th), 6^(th) or 7^(th) deciles (0.5, 0.6 or0.7 quantiles)) had greater clinical response rate differences fromplacebo (irrespective of which of the three different clinical responsemeasurements was used) compared to the LCN2 low subjects (including,e.g. subjects with LCN2 levels at the 1^(st) or 2^(nd) deciles (0.1 or0.2 quantiles)). These results further demonstrate the surprising andunexpected predictive value of high or elevated LCN2 serum levels inidentifying patients having an IL23-mediated disease or disorderresponsive to treatment with an IL23 antagonist (including, e.g., ananti-IL23 antibody or fragment thereof such as MEDI2070).

Finally, FIGS. 7, 8, and 9 and TABLE 6 also show that CD patientstreated with MEDI2070 having increasingly lower levels of baseline CCL20achieved higher clinical response rates (as measured using any of thethree different clinical response measurements shown in FIGS. 7, 8 and9) at week 8 compared to placebo, supporting that MEDI2070 inducedbetter clinical responses in patients with low baseline CCL20 serumlevels. Notably, CCL20 low subjects (including, e.g., subjects withCCL20 levels at the 1^(st), 2^(nd), 3^(rd), or 4^(th) deciles (0.1, 0.2,0.3 or 0.4 quantiles)) had greater clinical response rate differencesfrom placebo (irrespective of which of the three different clinicalresponse measurements was used) compared to the CCL20 high subjects(including, e.g. subjects with CCL20 levels at the 7^(th) or 8^(th)deciles (0.7 or 0.8 quantiles)). These results further demonstrate thepredictive value of low or decreased CCL20 serum levels in identifyingpatients having an IL23-mediated disease or disorder responsive totreatment with an IL23 antagonist (including, e.g., an anti-IL23antibody or fragment thereof such as MEDI2070).

For IL22 or LCN2, the relationship between increasing clinical responserates and increasing baseline biomarker levels was not always as evidentin patients in the 8^(th), 9^(th) or 10^(th) deciles. At these IL22 orLCN2 levels too few, if any, patients treated with MEDI2070 or placebowere available for analysis. See, e.g., FIGS. 7, 8, and 9; TABLES 4 and5. However, given a larger sample size, increased clinical responserates for patients identified as having IL22 or LCN2 levels at the8^(th), 9^(th) or 10^(th) deciles are expected.

Taken together, these results support that high IL22 serum levels and/orhigh LCN2 serum levels and/or low CCL20 (including, e.g., the medianbaseline IL22 and/or LCN2 serum levels identified in the study and/orserum IL22 levels between 7.9 pg/mL and 31.4 pg/mL and/or serum LCN2levels between 142.8 ng/mL and 261.1 ng/mL and/or serum CCL20 levelsbetween 4.9 pg/mL and 22.6 pg/mL) can be used to identify a patienthaving an IL23-mediated disease or disorder suitable for treatment withan IL23 antagonist (including, e.g., an anti-IL23 antibody or fragmentthereof such as MEDI2070).

TABLE 4 Subject counts and CDAI Response Rate at Week 8 by DecileBaseline IL22 Levels. MEDI2070 Placebo MEDI2070 Placebo BaselineMEDI2070 Non- Placebo Non- CDAI CDAI IL-22 responders respondersresponders responders Response Response Decile (pg/mL) (# subjects) (#subjects) (# subjects) (# subjects) rate (W8) rate (W8) 0 1 28 28 16 440.5000 0.2667 1 1 28 28 16 44 0.5000 0.2667 2 7.9 25 22 10 36 0.53190.2174 3 11.3 24 18 9 30 0.5714 0.2308 4 12.7 21 15 9 25 0.5833 0.2647 515.6 20 10 7 21 0.6667 0.2500 6 19.6 18 8 5 16 0.6923 0.2381 7 23.1 14 55 11 0.7368 0.3125 8 31.4 9 5 3 7 0.6429 0.3000 9 46.8 3 4 2 3 0.42860.4000 10 711 0 1 0 0

TABLE 5 Subject counts and CDAI Response Rate at Week 8 by DecileBaseline LCN2 Levels. MEDI2070 Placebo MEDI2070 Placebo BaselineMEDI2070 Non- Placebo Non- CDAI CDAI LCN2 Responders respondersResponders responders Response response Decile (ng/mL) (# subjects) (#subjects) (# subjects) (# subjects) rate (W8) rate (W8) 0 77.7 28 22 1535 0.5600 0.3000 1 142.8 27 19 13 31 0.5870 0.2955 2 163.6 23 16 12 290.5897 0.2927 3 184.3 21 14 12 23 0.6000 0.3429 4 201.3 20 14 6 200.5882 0.2308 5 214.6 18 12 4 16 0.6000 0.2000 6 233.4 16 8 3 13 0.66670.1875 7 261.1 14 6 3 7 0.7000 0.3000 8 294.8 10 4 2 4 0.7143 0.3333 9326.6 5 2 1 2 0.7143 0.3333 10 434.0 0 0 1 0

TABLE 6 Subject counts and CDAI Response Rate at Week 8 by DecileBaseline CCL20 Levels. MEDI2070 Placebo MEDI2070 Placebo BaselineMEDI2070 Non- Placebo Non- CDAI CDAI CCL20 Responders respondersResponders responders Response response Decile (pg/mL) (# subjects) (#subjects) (# subjects) (# subjects) rate (W8) rate (W8) 0 4.9 28 22 1535 0.56 0.3 1 11.88 26 20 14 30 0.5652 0.3182 2 14.58 25 18 13 24 0.58140.3514 3 16.37 20 17 12 21 0.5405 0.3636 4 20.8 16 15 11 19 0.51610.3667 5 22.65 13 12 9 16 0.52 0.36 6 26.58 10 10 8 12 0.5 0.4 7 32.1 77 8 9 0.5 0.4706 8 39.76 5 4 6 5 0.5556 0.5455 9 54.57 3 1 4 2 0.750.6667 10 224.1 1 0 0 0

TABLE 7 CDAI-100 response rate differential (defined as the differencein the percentage (%) of subjects achieving a CDAI-100 response betweentreatment and placebo) and CDAI remission rate differential (defined asthe difference in the percentage (%) of subjects achieving a total CDAIscore of below 150 points between treatment and placebo) for varioussubgroups. MEDI2070 CDAI MED12070 CDAI Response Rate Remission RateMinus Placebo Minus Placebo CDAI Response CDAI Remission Population NRate at Week 8 Rate at Week 8 mITT N = 119 20.8% 12.2% IL22 >= 11.3pg/mL N = 81 31.5% 18.3% IL22 >= 15.6 pg/mL N = 58 38.2% 29.2% CRP >= 5N = 85 30.8% 19.8% CRP >= 5 + IL22 >= N = 62 44.4% 26.9% 11.3 pg/mL

As noted previously, IL23-induced IL22 induces cells to secrete LCN2.Thus, the observation that baseline serum levels of two separate IL23pathway members (i.e. elevated IL22 or LCN2) each were predictive ofpatient clinical response to MEDI2070 (e.g. CDAI-100 Response Rate atweek 8) strongly suggests that other IL23 pathway biomarkers may alsopredict an increased likelihood of clinical effect in response totreatment with MEDI2070. Accordingly, to the extent that serum baselinelevels of IL22 and/or LCN2 or any other IL23 pathway analyte (including,e.g., CCL20, IL17F, IL17A/F, IL23R, IL12B, IL6, IL21, TNF, CCR6, CCL22,IL1R1, IFN-γ, S100A12, DEFB-2, DEFB-4, Ill, SERPINB3, PI3/Elafin, LL37,RORγ, RORγT, IL26, S100A7, DEFB103B, or GM-CSF) reflects increased IL23axis activity, a patient determined to have increased IL23 pathwayactivity (as determined by measuring one or more IL23 pathwaybiomarkers) may be more likely to benefit from treatment with an IL23antagonist (including, e.g., an anti-IL23 antibody or an antigen-bindingfragment thereof such as MEDI2070).

While the present invention has been described in terms of specificaspects, it is understood that variations and modifications will occurto those skilled in the art. Accordingly, only such limitations asappear in the claims should be placed on the invention.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary aspects, but should be defined onlyin accordance with the following claims and their equivalents.

The foregoing description of the specific aspects will so fully revealthe general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific aspects, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed aspects, based on the teaching and guidance presented herein.It is to be understood that the phraseology or terminology herein is forthe purpose of description and not of limitation, such that theterminology or phraseology of the present specification is to beinterpreted by the skilled artisan in light of the teachings andguidance.

What is claimed is:
 1. A method of treating an inflammatory boweldisease in a patient, comprising administering an IL23 antagonist to apatient if the patient is determined to have (i) a lower or decreasedlevel of CCL20 in one or more samples taken from the patient compared toa predetermined CCL20 threshold level, or compared to a CCL20 level inone or more control samples.
 2. A method of treating a patient having aninflammatory bowel disease comprising suspending the administration ofan IL23 antagonist to a patient if the patient is determined to have ahigher or increased level of CCL20 in one or more samples taken from thepatient compared to a predetermined CCL20 threshold level, or comparedto a CCL20 level in one or more control samples.
 3. A method of treatingan inflammatory bowel disease in a patient, wherein the patient failed,was non-responsive or intolerant to treatment with an anti-TNF agentcomprising administering an IL23 antagonist to the patient if thepatient is determined to have a lower or decreased level of CCL20 in oneor more samples taken from the patient compared to a predetermined CCL20threshold level, or compared to a CCL20 level in one or more controlsamples.
 4. The method according to any one of claims 1 to 3, furthercomprising measuring the level of CCL20 in one or more of the samplesobtained from the patient or instructing a clinical laboratory orhealthcare provider to measure the level of CCL20 in the sample and/orsubmitting the one or more samples obtained from the patient to aclinical laboratory or healthcare provider to measure the level of CCL20in the sample.
 5. The method according to any one of claims 1 to 3,wherein the IL23 antagonist is an anti-IL23 antibody or antigen-bindingfragment thereof.
 6. The method of claim 5, wherein the anti-IL23antibody or antigen-binding fragment thereof binds to the p19 subunit ofIL23 (SEQ ID NO: 15), to the p40 subunit of IL23 (SEQ ID NO: 16), orboth.
 7. The method according to claim 5, wherein the anti-IL23 antibodyor antigen-binding fragment thereof comprises ustekinumab, briakinumab,guselkumab, BI-655066, tildrakinumab, LY-3074828, or an antigen-bindingfragment thereof.
 8. The method according to claim 5, wherein theanti-IL23 antibody or antigen-binding fragment thereof comprises (i) aheavy chain variable region (VH) comprising or consisting of SEQ ID NO:7 and a light chain variable region (VL) comprising or consisting of SEQID NO: 8, or (ii) a heavy chain variable region (VH) comprising orconsisting of SEQ ID NO: 45 and a light chain variable region (VL)comprising or consisting of SEQ ID NO:
 46. 9. The method according toclaim 5, wherein the anti-IL23 antibody or antigen-binding fragmentthereof comprises a heavy chain variable region comprising SEQ IDNOS:33, 34, and 35 and a light chain variable region comprising SEQ IDNOS:36, 37 and 38, or a heavy chain variable region comprising SEQ IDNOS:47, 48 and 49 and a light chain variable region comprising SEQ IDNOS:50, 51 and
 52. 10. The method according to claim 9, wherein theantibody is administered at a fixed dose.
 11. The method according toclaim 5, wherein the antibody is administered at a fixed dose.
 12. Themethod according to claim 11, wherein the fixed dose is between 10 and1000 mg/dose.
 13. The method according to claim 11, wherein the fixeddose is about 210 mg/dose or about 700 mg/dose.
 14. The method accordingto any one of claims 1 to 3 wherein the patient has been treated before,during, after, or alternatively to the administration of an IL23antagonist or anti-IL23 antibody or antigen-binding fragment with one ormore additional therapies for the treatment of the inflammatory boweldisease.
 15. The method according to any one of claims 1 to 3, whereinthe one or more samples taken from the patient and/or the one or morecontrol samples are one or more selected from the group consisting of:whole blood, blood serum, plasma, saliva, sputum, bronchoalveolar lavagefluid, cerebrospinal fluid, pleural fluid, pericardial fluid, ascites,synovial fluid, epithelial cells, urine, stool, skin, tissue biopsy, ora combination thereof.
 16. The method according to any one of claims 1to 3, wherein the one or more control samples are (i) a sample orsamples obtained from normal healthy individuals; (ii) a sample orsamples obtained from patients with a non-IL23-mediated disease; or(iii) a combination thereof.
 17. The method according to any one ofclaims 1 to 3, wherein the patient's level of CCL20 is measured in animmunoassay.
 18. The method according to any one of claims 1 to 3,further comprising determining the level of one or more IL23 PathwayBiomarkers selected from the group consisting of IL22, LCN2, ID17F,IL17A/F, IL23R, ID12B, IL6, IL21, TNF, CCR6, CCL22, ILR1, IFN-γ,S100A12, DEFB-2, DEFB-4, IL1, SERPINB3, PI3/Elafin, LL37, RORγ, RORγT,IL26, S100A7, DEFB103B, and GM-CSF.
 19. The method according to any oneof claims 1 to 3, wherein the predetermined threshold level of CCL20 isselected from the group consisting of: (a) about the mean level ofCCL20; (b) about the median level of CCL20; and (c) about the 1^(st),2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th), 8^(th), or 9^(th) decilebaseline level of CCL20 of 4.9 pg/ml, 11.88 pg/ml, 14.58 pg/ml, 16.37pg/ml, 20.8 pg/ml, 22.65 pg/ml, 26.58 pg/ml, 32.1 pg/ml 39.76 pg/ml, or54.57 pg/ml, respectively; as measured in the serum using an immunoassayfrom a plurality of normal healthy patients, patients with anon-IL23-mediated disease, and/or patients with an inflammatory boweldisease.
 20. The method according to any one of claims 1 to 3, whereinthe inflammatory bowel disease or disorder is selected from the groupconsisting of: Crohn's disease, ulcerative colitis (UC), and celiacdisease.
 21. The method according to any one of claims 1 to 3, whereinthe predetermined CCL20 threshold level is at least about 5 pg/mL to atleast about 55 pg/mL as measured using an immunoassay.
 22. The methodaccording to claim 21, wherein the predetermined CCL20 threshold levelis about 22.6 pg/mL as measured using an immunoassay.
 23. The methodaccording to claim 22, wherein administration of the IL23 antagonist oranti-IL23 antibody or antigen-binding fragment thereof results in aCrohn's Disease Activity Index (CDAI) response score reduction of atleast 100 points and/or a reduction in the total CDAI score to below 150points after first administering the anti-IL23 antibody orantigen-binding fragment thereof.
 24. The method according to claim 23,wherein the CDAI response score reduction of at least 100 points orreduction in the total CDAI score to below 150 points occurs within 1,2, 4, 8, 12, 16 or 24 weeks or later after first administering the IL23antagonist or anti-IL23 antibody or antigen-binding fragment thereof.25. The method according to claim 21, wherein administration of the IL23antagonist or anti-IL23 antibody or antigen-binding fragment thereofresults in a Crohn's Disease Activity Index (CDAI) response scorereduction of at least 100 points and/or a reduction in the total CDAIscore to below 150 points after first administering the anti-IL23antibody or antigen-binding fragment thereof.
 26. The method accordingto claim 25, wherein the CDAI response score reduction of at least 100points or reduction in the total CDAI score to below 150 points occurswithin 1, 2, 4, 8, 12, 16 or 24 weeks or later after first administeringthe IL23 antagonist or anti-IL23 antibody or antigen-binding fragmentthereof.
 27. The method according to claim 1, wherein the inflammatorybowel disease is Crohn's disease, UC or Celiac Disease.
 28. The methodaccording to claim 27, wherein the patient is determined to have a levelof CRP≥5 mg/L and/or a level of FCP≥250 μg/g, a level of FCP≥200 μg/g, alevel of FCP≥150 μg/g, a level of FCP≥100 μg/g, or a level of FCP atleast about 100 μg/g to at least about 250 μg/g in one or more samplestaken from the patient.
 29. The method according to claim 28, whereinadministration of the IL23 antagonist or anti-IL23 antibody orantigen-binding fragment thereof results in a Crohn's Disease ActivityIndex (CDAI) response score reduction of at least 100 points and/or areduction in the total CDAI score to below 150 points after firstadministering the anti-IL23 antibody or antigen-binding fragmentthereof.
 30. The method according to claim 29, wherein the CDAI responsescore reduction of at least 100 points or reduction in the total CDAIscore to below 150 points occurs within 1, 2, 4, 8, 12, 16 or 24 weeksor later after first administering the IL23 antagonist or anti-IL23antibody or antigen-binding fragment thereof.
 31. The method accordingto claim 27, wherein administration of the IL23 antagonist or anti-IL23antibody or antigen-binding fragment thereof results in a Crohn'sDisease Activity Index (CDAI) response score reduction of at least 100points and/or a reduction in the total CDAI score to below 150 pointsafter first administering the anti-IL23 antibody or antigen-bindingfragment thereof.
 32. The method according to claim 31, wherein the CDAIresponse score reduction of at least 100 points or reduction in thetotal CDAI score to below 150 points occurs within 1, 2, 4, 8, 12, 16 or24 weeks or later after first administering the IL23 antagonist oranti-IL23 antibody or antigen-binding fragment thereof.